text:
Next: Garbage Collection, Previous: Building Emacs, Up: GNU Emacs Internals [Contents][Index]
Emacs Lisp uses two kinds of storage for user-created Lisp objects: normal storage and pure storage. Normal storage is where all the new data created during an Emacs session are kept (see Garbage Collection). Pure storage is used for certain data in the preloaded standard Lisp files—data that should never change during actual use of Emacs.
Pure storage is allocated only while temacs
is loading the
standard preloaded Lisp libraries. In the file emacs, it is
marked as read-only (on operating systems that permit this), so that
the memory space can be shared by all the Emacs jobs running on the
machine at once. Pure storage is not expandable; a fixed amount is
allocated when Emacs is compiled, and if that is not sufficient for
the preloaded libraries, temacs allocates dynamic memory for
the part that didn't fit. The resulting image will work, but garbage
collection (see Garbage Collection) is disabled in this situation,
causing a memory leak. Such an overflow normally won't happen unless
you try to preload additional libraries or add features to the
standard ones. Emacs will display a warning about the overflow when
it starts. If this happens, you should increase the compilation
parameter SYSTEM_PURESIZE_EXTRA
in the file
src/puresize.h and rebuild Emacs.
This function makes a copy in pure storage of object, and returns it. It copies a string by simply making a new string with the same characters, but without text properties, in pure storage. It recursively copies the contents of vectors and cons cells. It does not make copies of other objects such as symbols, but just returns them unchanged. It signals an error if asked to copy markers.
This function is a no-op except while Emacs is being built and dumped; it is usually called only in preloaded Lisp files.
The value of this variable is the number of bytes of pure storage allocated so far. Typically, in a dumped Emacs, this number is very close to the total amount of pure storage available—if it were not, we would preallocate less.
This variable determines whether defun
should make a copy of the
function definition in pure storage. If it is non-nil
, then the
function definition is copied into pure storage.
This flag is t
while loading all of the basic functions for
building Emacs initially (allowing those functions to be shareable and
non-collectible). Dumping Emacs as an executable always writes
in this variable, regardless of the value it actually has
before and after dumping.
You should not change this flag in a running Emacs.
Next: Garbage Collection, Previous: Building Emacs, Up: GNU Emacs Internals [Contents][Index]


text: Did you have yet another slow start this Monday morning? It happens to the best of us, but that doesn't mean it has to stop you from having a productive and energetic day once you arrive at the office. Here are a few tips on how to build and maintain positive energy throughout the day.… Read More

subset: commoncrawl


text:
Meulen, Quiryn van der (d. ca. 1600)
Quiryn van der Meulen (Crijn, Krijn, Cryn Vermeulen), was the elder of the Danzig congregation following Dirk Philips in the last decades of the 16th century. His letters that were preserved in the records of the Heubuden church were signed by his Flemish name Cryn Vermoolen. In the disputes on the ban which divided the Dutch Mennonites into two camps he was on the side of the merciful use of the ban. (H. G. Mannhardt's judgment, p. 45, concerning Cryn's severe rule in the church is based on error.) This generosity led to tensions within the Danzig congregation and beyond in the West Prussian churches, who on the basis of a correspondence with the leaders of the Flemish wing in Holland placed the milder Frisians under the ban.
In 1580 Cryn still had the confidence of all the Mennonites of West Prussia. He was one of several delegates sent by the West Prussian churches to the conference of elders in Haarlem and Emden. He was saddened by what he heard about the strife between the Frisians and Flemish. "I confess that in Haarlem I showed my feeling, which was inclined to peace, though in my worry I received no help." He requested that men be sent to Danzig to teach the brethren that which he himself could not understand, and declared that if he was proved to be in error he was willing to be dismissed from his eldership. When these men came to Danzig, he expressed himself freely on his concern upon their request that he do so.—The name "Bekommerden" (i.e., Concerned Ones) for the lenient wing of the Frisians is derived from their concern or worry about the severe application of the ban.
Van der Meulen's frankness aroused offense in Danzig. He was accused of having destroyed the peace of the congregation. On the advice of the Haarlem church representatives of the three Frisian congregations of Montau, Elbing, and Olde Tooren (Thorn), a total of 35 brethren met and confronted Cryn and his friend Hans van Schwindern with their charge of guilt, and presented to them a confession they had worked out in advance. It contained six charges: (1) Cryn and Hans had criticized the elders in the west and had thereby created disturbance in the brotherhood. (2) In spite of the wishes of the West Prussians that they attend a conference of elders in Holland they had not complied, but had rather burdened the Danzig church with "weaknesses and liberties." (3) Cryn had promised in Haarlem that he would remain quiet, but did not do so. (4) Cryn and Hans had censured the deceased Dirk Philips (for his strict conception of the ban) against the will of the congregation. (5) Cryn and all those who thought as he did did not rebaptize those who joined the brotherhood from some other branch, and did not apply the strict ban. (6) In addition to this charge, there was his "peculiar understanding" of the truth.
Cryn defended himself in a letter. The tone and content of this letter show his superiority. The charges had no justification, and as to his view of the ban, he maintained, "We would prefer to have matters go according to Christ's teaching and take their course after fraternal discussion." His personality and the strength of his conviction caused many of his opponents to waver. The confession of guilt was probably read to the 35 delegates of the three churches in Danzig in the home of Harmen Borgerlinck (25 September 1586), but they did not yet dare to pass judgment on Cryn and his supporters. But when they arrived in Holland and there received sanction the committee met once more in Danzig early in 1588; however, the representatives from Elbing and Thorn left at once. And so Hilchen Smit (Schmidt), the elder of the Montau congregation, with his delegates and 55 brethren from the Danzig congregation, was obliged to carry out the expulsion of Cryn and Hans van Schwindern. The act was direful. The cleavage between the Frisians and the Flemish, which was bridged over by Cryn and men like him, now became open. As early as March a protest was made to the leaders by the Elbing congregation, disapproving the hard course taken by the Montau group. They begged for patience and long-suffering toward Cryn.
Cryn continued to live in Danzig as an ordinary member of the congregation. Alenson's Tegenbericht (Bibliotheca Reformatoria Neerlandica VII, 190) reports that in a colloquy with Wolff Uohll, who had come to Danzig from Moravia with Jan Gerrits, Cryn upheld Menno's view of the Incarnation. He applied his energy to writing. The [[Amsterdam Mennonite Library (Bibliotheek en Archief van de Vereenigde Doopsgezinde Gemeente te Amsterdam)|Amsterdam Mennonite Library]] and the Danzig Church Library each have a copy of a beautiful Schottland Bible which he had printed at his own expense. The foreword contains the statement, "Men vindse te koop bij Krijn Vermeulen de jonghe, Cramer, woonende opte lege zydt van Schotlandt bij Danswick 1598" (To be bought of Krijn Vermeulen, Jr., merchant, living on the lower side of Schottland at Danzig, 1598). (Mannhardt, op. cit.)
Krijn van Vermeulen is not to be confused with the Danzig Mennonite alchemist Krijn van Mollem.
Hege, Christian and Christian Neff. Mennonitisches Lexikon, 4 vols. Frankfurt & Weierhof: Hege; Karlsruhe: Schneider, 1913-1967: v. III, 118 f.
Mannhardt, H. G. Die Danziger Mennonitengemeinde. Danzig, 1919.
Succssio Anabaptistica and Alenson's Tegenbericht, Cramer, Samuel and Fredrik Pijper. Bibliotheca Reformatoria Neerlandica, 10 vols. The Hague: M. Nijhoff, 1903-1914: VII, 65 and 190.
Author(s) | Horst Quiring |
Date Published | 1957 |
Cite This Article
MLA style
Quiring, Horst. "Meulen, Quiryn van der (d. ca. 1600)." Global Anabaptist Mennonite Encyclopedia Online. 1957. Web. 25 Jul 2024. https://gameo.org/index.php?title=Meulen,_Quiryn_van_der_(d._ca._1600)&oldid=164125.
APA style
Quiring, Horst. (1957). Meulen, Quiryn van der (d. ca. 1600). Global Anabaptist Mennonite Encyclopedia Online. Retrieved 25 July 2024, from https://gameo.org/index.php?title=Meulen,_Quiryn_van_der_(d._ca._1600)&oldid=164125.
Adapted by permission of Herald Press, Harrisonburg, Virginia, from Mennonite Encyclopedia, Vol. 3, pp. 661-662. All rights reserved.
©1996-2024 by the Global Anabaptist Mennonite Encyclopedia Online. All rights reserved.


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Four years ago, when Barack Obama was running against John McCain for the Presidency and when the climate crisis was not as severe as it has become, both Obama and McCain were in agreement that 1) it existed and 2) action should be taken on it. As importantly, they both spoke about this issue throughout the campaign.
Obama's program back then, put forward in early August 2008, projected 1 million plug-in hybrid cars by 2015, a commitment to energy efficiency, investing in an upgrade of the national utility grid, weatherizing one million homes annually, a 100% auction—with no freebies for polluters–of carbon emission permits, and a goal of 5 million new green jobs. He consistently incorporated language in his speeches for the US to advance wind and solar, to "heal the planet."
Compared to then-President George Bush, these were important, if limited, steps in the right direction, and Obama and the Democrats have made efforts, with some success, to move most of these and other clean energy programs forward.
But when it comes to talking about the much-worse, deepening climate crisis in 2012, Obama has essentially censored himself. It is stunning, and deeply disturbing, that this is happening, given events just this year like record heat and widespread drought and a new record for Arctic ice melt. Some climate scientists who have been studying the Arctic for many years believe that it is possible that before the next Presidential election in 2016, the Arctic could be ice-free during the last month of summer.
What is baffling about Obama's silence is that polling shows consistently that taking action on climate is a winner politically. A poll of likely voters just last month conducted by Yale and George Mason University found these striking results:
"Most undecided likely voters (80%) believe that global warming is happening, while only 3% say it is not happening – which is very similar to likely Obama voters (86% and 4%, respectively).
"Two out of three Undecideds (65%) say that if global warming is happening, it is mostly human caused, the same as likely Obama voters (65%).
"Though few likely voters say global warming is the "single-most important" issue to them in this election, majorities of both likely Obama voters (75%) and Undecideds (61%) say it will be one of several important issues determining their vote for President.
"Undecideds as well as likely Obama voters say that President Obama (64% and 61% respectively) and Congress (72% and 78%) should be "doing more" about global warming."
For several months many climate and environmental groups have tried to break the silence on climate in the Presidential election campaign, so far without success. An on-line petition effort, www.climatesilence.org, was recently launched. Over 150,000 people signed a petition to Jim Lehrer to get him to ask a question on climate in the first debate, without success.
But in retrospect, I wonder what would have happened even if Lehrer had asked such a question. Given Obama's, and Romney's, respective first debate performances, there's a good chance they both would have said little of substance, done all they could to keep this from becoming a major issue.
Obama's debate performance, as indicated by polls released over the last several days, show that Obama has pretty much lost the lead he had been slowly developing prior to the debate. All signs point to a very close election that, as of now, could go either way.
In that context, and given the results of the Yale/George Mason and other prior polls, it sure seems like it would be to Obama's benefit that he regain his voice on climate. It's true that, like debating, this is something that he's not used to. He hasn't been talking about climate for years, literally. Ever since the failed Copenhagen climate conference in late 2009, Obama has rarely mentioned the issue, and when he has said something that's all that it has been, a mention.
But our current President does have a history of coming off the ropes with strength and passion when it has to do with his political standing or possible election defeat. If he and his campaign advisors finally realize that publicly speaking up for action to address the climate crisis is the right thing to do politically, maybe that will be what breaks the silence and, indeed, plays a key role in getting Obama elected.
It's a shame if this ends up being what causes it, but given our desperate climate reality, it's something to be welcomed. The planet, its people and all forms of life need a President who doesn't make jokes about but who has a vision, however cloudy, of a time when the "rise of the oceans begins to slow and our planet begins to heal."


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Child care costs continue to soar, with prices so high in some parts of the country that they exceed tuition at state colleges.
Last year, average center-based child care costs rose by nearly 3% nationwide, according to a report from the nonprofit Child Care Aware of America. Full-time care for an infant ranged from a high of $16,430 a year in Massachusetts to $4,863 in Mississippi. Meanwhile, center-based care for a four-year-old hit a high of $12,355 in Massachusetts and a low of $4,312 in Mississippi.
Why such huge price disparities? Blame it on differences in labor costs, state regulations and cost of living expenses, such as housing, food and utilities.
Related: How much will it cost to raise your child?
For example, Massachusetts has strict child care regulations that require one teacher for every three infants, compared to one teacher per five infants in Mississippi. Meanwhile, child care centers in New York City, among one of the most expensive places for child care in the country, pay significantly higher rents and also must meet strict state standards.
"In order to meet those (standards), it costs money," said Jessica Klos Shapiro, public policy and communications coordinator at the nonprofit Early Care & Learning Council, which advocates for families across New York state.
The centers are also grappling with ballooning operational costs, ranging from rising insurance costs to higher food prices, said Lynette Fraga, Child Care Aware's executive director.
As a result, child care costs grew by as much as eight times the rate of family incomes last year, the report said. And they continue to take a major chunk out of family budgets, often representing a household's largest monthly expense.
Center-based infant care for one child was greater than median rent payments in nearly half of the states, while fees for two children (an infant and a four-year-old) exceeded rent in all 50 states. And in nearly two-thirds of the country, average child care costs were greater than yearly tuition and fees at a four-year public college.
Related: 'How we afford our kids'
To determine affordability, Child Care Aware compared average child care costs to the state's median family income. Oregon was the least affordable state, with average care for an infant representing more than 18% of the median income for a married couple. New York, Minnesota, Massachusetts and Colorado rounded out the five least affordable states.
In recent years, Denver resident Liz Sagaser and her husband have spent as much as $1,400 a month on full-time child care for their two children.
Their 5-year-old daughter started public kindergarten this fall, and their 3-year-old son is in public preschool. But they still spend around $1,000 a month on education and child care, which takes up around 13% of their annual income.
"That's a mortgage for a lot of people, and that's just the child care costs," she said. "That's a huge portion of our living expenses."
Related: How much is child care in your state?
With nearly 11 million children under the age of five needing child care services and not enough government subsidies to go around, working parents are forced to make difficult choices, said Fraga. Many end up choosing cheaper non-licensed options, which don't have to meet health and safety standards.
"The big question mark is: 'Are children safe in unregulated care?'" she said, adding that federal budget and sequester cuts "threaten to make an already strained situation worse."
Least affordable states for child care
State | Avg. cost of center-based infant care | Median income for married couple | Percent of Income |
Oregon | $13,452 | $72,226 | 18.60% |
New York | $14,939 | $90,725 | 16.50% |
Minnesota | $13,876 | $89,608 | 15.50% |
Massachusetts | $16,430 | $109,090 | 15.10% |
Colorado | $12,736 | $85,137 | 15.00% |
California | $12,068 | $81,484 | 14.80% |
Illinois | $12,697 | $85,715 | 14.80% |
Hawaii | $12,473 | $86,699 | 14.40% |
Washington | $12,108 | $84,464 | 14.30% |
Kansas | $10,518 | $77,034 | 13.70% |


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As a pet owner, you will soon learn that you can't always be home to take care of your pet. Whether your travel destination doesn't allow pets or you simply work eight hours per day, hiring a pet sitter may seem fitting. Before hiring someone to look after your pet, always check their references and verify whether the sitter is a member of the National Association of Professional Pet Sitters or Pet Sitters International. Both of these institutions pride themselves on providing superior knowledge about professional pet care so being affiliated with either is notable. Check that the pet sitter is insured and licensed in pet CPR. When you have finally found a pet sitter who meets your requirements, be sure there is a signed agreement outlining your pet's care, including how frequently the pet sitter will visit.
Benefits of hiring a pet sitter
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Marine Corps
Exceptional Family Member Program / Family Support
O'Bannon Ave. & Iwo Jima Ave.
McTureous Hall, Bldg 218
Barstow, CA 92311-5050
Contact information for key programs and services at this installation.
The Education Directory for Children with Special Needs was developed to support military families who have children (birth through 21) with special educational needs. Military families can consult the directory for information on special-needs issues and listings of national and local resources for early intervention (for infants and toddlers) and special education services (for school-age children).
The Education Directory for Children with Special Needs provides information to help you make informed educational decisions for your child with special needs. The directory provides information about:
The directory has two parts:
Release 2024.03.06.1


text:
If you're reading this guide, it's likely that you want to improve your memory or focus. Neuro enhancers or smart drugs are often a popular choice among students, learners and adults. The truth is that everyone wants to have a better memory, and if you look this up, you'll likely find people talking about nootropics.
But what in the world is a nootropic?
Nootropics are essentially supplements that you will take to boost your memory power. And if you're trying to define nootropics, you would classify these as: memory supplements or foods that improve mental function.
Defining mental function is dependent on the nootropic that is consumed. In most cases, these supplements will:
- Improve memory
- Improve motivation
- Improve attention span
Many of the nootropics that you will find on the market are used for people with memory -related diseases (think Alzheimer's disease or dementia).
How Do They Work?
The best nootropics work much like your multivitamin that you take in the morning.
These supplements will accumulate over time in the body, like vitamins. When you first begin taking them, you may not notice an immediate difference. This is because your body is still lacking the essential vitamins that it needs to operate at 100% capacity.
Over time, your body will begin to use these supplements to start changing chemically.
It's a rather easy concept to understand. When you first begin going to the gym, you may not notice immediate results. You may notice that you're sore, or your strength may increase slowly, but if you were to keep at it for two or three months, you'll notice that your body begins to change. You have more stamina, you may be trimmer, and you'll look better. This is exactly what happens when you take a nootropic, but it happens internally with the brain.
We'll be discussing various types of nootropics, and how each one works individually.
Since each has its own unique chemical makeup, minerals and nutrients, they will all work differently.
Essentially, you'll:
- Take a supplement (1 to 2 times per day)
- Start to notice an improvement
It's important to note that the best nootropics will need to be taken routinely to experience results. This is not something that you can take for just a few weeks and keep reaping the benefits when you stop taking the pills. It doesn't work this way.
Nootropics and Academia
What better place to try a memory enhancer than an educational institute? There have been several studies on nootropics, which we will discuss now.
- One study shows that 0.7% to 4.5% of students in Germany have used cognitive enhancers.
- Another study showed that 5% to 35% of college students have used performance enhancement drugs, such like those found in ADHD medications to boost their academics.
A major issue is that it is rather difficult to judge or analyze exactly how much more memory retention a person will have after taking a supplement. There may be a chance that the material is rather interesting to the person, or is a topic they know, so it is very difficult to judge the effectiveness of nootropics without looking at a particular type.
If you want an example of this, look no further than bacopa. A study on this memory supplement explains how it works scientifically to change the chemical makeup of the brain, and it was shown that this supplement is very beneficial for the retention of learned material. The study also showed that the supplement can reduce the rate of short-term memory loss or forgetting.
Understanding Brain Health Supplements
Brain supplements target various different parts of the brain and the way that we remember information. For example, one supplement may be able to help you increase your cognitive function, while another may boost your memory retention.
Many people will take nootropics in what is called a "stack."
What is a nootropic stack?
A stack would consist of several different brain supplements that all aid in either:
- Memory enhancement
- Attention enhancement
- Energy boosting
So, you may take the best memory supplements along with cognitive enhancers, caffeine and other supplements to all work in conjunction to boost your memory.
The best part is that the best nootropics will include an essential "stack."
What About Side Effects?
Remember, every nootropic is different, so the side effects experienced will be different depending on which supplement you're taking. And not everyone experiences the same side effects. You'll find that the side effects that you may experience are mild, and they might include:
- Jitters
- Headaches
- Upset stomach
- Nausea
I've personally found that I'm a little sensitive to some nootropics – I get headaches after two months of usage.
I'm not sure if this is common, but I found that taking them for two months and then stopping for a week or two before going back to them solves my headache issues. Upset stomach, jitters and nausea are something I personally have never experienced. Jitters are normally experienced due to caffeine within the product, so if you're sensitive to caffeine, you may want to keep this in mind.
Nootropics Categories or Classifications
If you're looking for the best nootropic for memory, you'll find that this truly depends on a person's preference. Most people will start seeing results within hours or days, depending on the product, but full results are not experienced immediately. These are not like magic diet pills that give your results in days, you need to be consistent to get results.
There are several different categorizations that you want to know about:
These are the typical supplements that you'll find. Normally, these are herbs or ingredients that you purchase to enhance your brain function. Common nootropics in this category include:
- Bacopa Monnieri: One of the most popular supplements on the market. Further research is needed, but the two in-depth studies that have been done on this ingredient show that it does have the potential to enhance your memory.
- Panax Ginseng: This is an ingredient that has been used for centuries in Asia. Many Asian studies point to this increasing memory, increasing speed when performing tasks and an improvement in understanding very difficult material.
- Salvia Officinalis: Not widely studied, this is still one of the most popular brain enhancers.
- Gingko Biloba: Several reviews have been done on ginkgo biloba. One study showed that this ingredient does offer significant help with selective attention, long-term memory and executive processes.
Racetams are what you'll find with over-the-counter products. Many of the best nootropic products will fall into this category. Essentially, racetams are packed with several ingredients in an effort to boost your memory.
The best racetams include:
- Pramiracetam: A supplement that stimulates the central nervous system. Discovered in the late 1970s, this product has been used in the treatment of memory and attention deficits for aging people.
- Aniracetam: A nootropic that is considered to be more potent than piracetam. This product has cognitive enhancing effects, and has helped people with Alzheimer's disease. It's important to note that this is considered a prescription drug in Europe, and it is not approved by the FDA.
- Noopept: An extremely popular nootropic. While technically not a racetam because of its chemical makeup, this product is often grouped into the same category. Several studies have been done on this product, and there have been notable improvements in memory and attention.
Our goal is to find the best supplement for memory, and there are a lot to choose from. There are several different over-the-counter products available, and most of them worked to a limited extent. As you can imagine, trying each of these products over time for one or two months is a difficult task considering that there are dozens of products available.
But knowledge is power right?
The best nootropics are available for an affordable price, and they have been tested time and time again. These supplements offer very few side effects, and have an amazing upside, with various studies done on several of the ingredients included. If you are to take a natural enhancer, I recommend looking at the ingredients themselves and doing your own research on each ingredient individually.
It's not common to find nootropics that are not in their own "stack" form already.
Results normally kick in immediately (30 minutes), and they will be able to boost your attention span. Energy improvements and attention span are almost immediately improved, while cognition and memory retention improve slightly over time before you really hit your maximum potential.


text:
Photomicrography under fluorescence illumination conditions presents a unique set of circumstances posing special problems for the microscopist. Exposure times are often exceedingly long (in some instances running from many seconds into several minutes), the specimen's fluorescence may fade during exposure, and totally black backgrounds often inadvertently signal light meters to suggest overexposure.
In addition, fluorescing specimens emit their own light, and particles residing above and below the desired plane of focus often radiate light causing blurring of the image details. Even though fluorescence images may appear to be bright when viewed through the microscope eyepieces (due to the human eye's exquisite sensitivity to light), they usually require lengthy exposure times in order to register a satisfactory image on film. Very long exposures complicate photomicrography of specimens stained with fluorescent dyes by increasing the likelihood of vibration and film reciprocity failure effects causing undesirable color shifts in the image.
While the basic problem with fluorescence photomicrography is often the paucity of light reaching the film, there are a number of steps that can be taken to improve image quality. Microscope objectives utilized for fluorescence should have as high a numerical aperture as possible, with high transmission values for the wavelengths of light being employed in the experiments. Because the light intensity (in reflected light fluorescence) varies as the fourth power of the numerical aperture, these objectives can significantly reduce exposure times in reflected light fluorescence microscopy. Light intensity also varies inversely as the square of the magnification, resulting in brighter images when the total magnification on film is held to a minimum. Reduction of magnification on the film plane is often assisted by the use of low magnification photoeyepieces (or projection lenses). In addition, when using a trinocular head, it is usually desirable to divert all light to the camera rather than have the light split (by means of a beamsplitter) between the viewing eyepieces and the camera. Because the image intensity also varies inversely as the square of the distance to the film plane, microscopes perform better then this distance is kept short. For this reason, and due to widespread availability, most fluorescence microscopy is recorded on 35 millimeter film (as opposed to larger format) when using traditional photomicrography to capture images. Modern low-noise temperature-controlled CCD digital cameras are being increasingly used to replace film as the medium of choice for fluorescence photomicrography.
Low light levels and the fading of fluorescent specimens are two of the primary difficulties to overcome in fluorescence photomicrography. Often, the dynamic nature of fluorescent preparations makes good photomicrography essential to capture a record of events occurring with the specimen, and often may reveal details not obvious when examining the specimen through the eyepieces. Long exposures, so commonly used in fluorescence, inevitably lead to reciprocity failure and fading of specimen fluorescence intensity during prolonged irradiation. In fact, fading of specific fluorescent features is often faster than that of the background, leading to a loss of image contrast.
All photographic emulsions (including black & white, color negative, and color transparency films) suffer from an effect known as reciprocity law failure when exposed to light for exceeding long or short time periods. The reciprocity law relates image density to exposure time and light intensity, such that there is a reciprocal relationship between illumination intensity in the microscope and the required exposure time for film. In effect, the law states that exposure time is inversely proportional to light intensity in order to maintain a constant film density. The lower the light intensity, the longer the necessary exposure time required to produce a satisfactory image. This inverse relationship holds true for most films if the proper exposure duration is between 1/500 of a second and 1/2 second.
In fluorescence microscopy, low light levels routinely demand exposures that exceed 1/2 second (often ranging from several seconds to several minutes). The reciprocity relationship no longer holds under these conditions, and the film will require additional exposure time to yield the proper image density. This phenomenon is called reciprocity failure, a term that only indicates that the linear relationship between exposure time and light intensity no longer holds and does not suggest a failure of the film emulsion in terms of performance.
A side effect of reciprocity law failure is that the already weak specimen fluorescence is further reduced by photobleaching of the specimen, and incorrect color reproduction often occurs due to increased exposure times. The series of images presented in Figure 2 are fluorescence photomicrographs illustrating antibody fluorochrome labeling with the popular reagent fluorescein-5-isothiocyanate (FITC) when imaged using an Olympus WIB long-pass fluorescence cube. The cells in the center (Figure 2(b)) were photographed using a corrected exposure time adjusted for reciprocity law failure and Kodak Wratten color compensating filters to remove an undesirable color shift. On the left (Figure 2(a)), the same viewfield shows the effect of photobleaching, or fading of the fluorescence activity due to prolonged exposure to the illumination source. Color shifts and reduced image intensity resulting from reciprocity law failure are evident in Figure 2(c).
Fluorescence Filter Spectra
Explore the overlap of fluorescence filter spectra and how changes in these filters determines the bandwidth of wavelengths passed through the various filter combinations.
Reciprocity failure can often be compensated simply by an increase in exposure time or processing conditions for black & white films, but this is not always the case with color negative and transparency films. Most color films have three color-sensitive dye layers, each of which has a slightly different characteristic curve position and slope resulting in varied responses to the reciprocity effect with the potential to cause undesirable color shifts or casts. Often, both exposure times and color balance filters must be adjusted to compensate for very long or short exposures when using color films.
Utilization of an objective having a very small numerical aperture (for instance, a 40x objective with NA = 0.65 as opposed to the same magnification objective having a NA of 0.85, 0.95, or 1.0) produces an image displaying insufficient brightness, which results in longer exposure times. The numerical aperture is a measure of the objective's light gathering ability, meaning that high numerical aperture objectives can capture a greater amount of the weak fluorescence emission produced by chromophores. Always use the highest available objective numerical aperture when doing fluorescence photomicrography. Using excessive objective and photoeyepiece magnifications will also result in reduced brightness. To compensate for this, always employ the lowest possible magnification photoeyepieces and the highest numerical aperture objectives at a given magnification. For example, a 60x plan apochromat objective having a numerical aperture of 0.95 (brightness index = 22.6) is preferable to a 60x plan fluorite objective of numerical aperture equal to 0.85 (brightness index = 14.5) even though the working distance of the fluorite objective is twice that of the apochromat. Furthermore, since high numerical aperture objectives have a shallower depth of field, their use may avoid contrast-reducing effects of out-of-focus emission from above or below the plane of focus. Likewise, a 2.5x photoeyepiece will provide more illumination than a 3.3x or 5x eyepiece. Laser scanning confocal microscopes are specifically designed to virtually eliminate out-of-focus light and give much clearer fluorescence pictures with somewhat better resolution.
Objective Brightness Values
Objective Magnification | Numerical Aperture (NA) | Objective Brightness Index |
10 | 0.25 | 3.9 |
10 | 0.30 | 8.1 |
10 | 0.45 | 41.0 |
10 | 0.50 | 62.5 |
20 | 0.40 | 6.4 |
20 | 0.50 | 15.6 |
20 | 0.75 | 79.1 |
40 | 0.65 | 11.1 |
40 | 0.75 | 19.8 |
40 | 0.95 | 50.9 |
40 | 1.00 | 62.5 |
40 | 1.30 | 178.5 |
60 | 0.85 | 14.5 |
60 | 0.95 | 22.6 |
60 | 1.40 | 106.7 |
100 | 1.25 | 24.4 |
100 | 1.40 | 38.4 |
Table 1
Typical fluorescence applications in fields such as cell biology and medical diagnostics are not only concerned with specimen brightness, but also the ratio of specific specimen fluorescence to the intrinsic background fluorescence. This is particularly important for single-molecule fluorescence events and other low light experiments where autofluorescence and/or internal reflection inside the objective produces vast differences in the ability to image small structures with low fluorescence quantum yields. To maximize the signal-to-noise ratio (intensity of the specimen fluorescence above the background) in objectives designed for fluorescence microscopy, manufacturers use quartz and other special glass formulations that have high transmission values throughout the spectrum from infrared to the ultraviolet. These objectives are extremely low in autofluorescence and utilize special optical cements and anti-reflection coatings designed to operate through an extended range of fluorescence excitation wavelengths. Higher numerical apertures coupled to advanced glass formulations provide modern fluorescence objectives with the ability to excite specimens at wavelengths down to 340 nanometers with much higher transmission values in order to obtain a brighter fluorescence image.
A comparison of nominal objective brightness values for common magnifications and numerical apertures is presented in Table 1. These numbers were calculated according to the following equation:
where NA is the objective numerical aperture and the Transmission Ratio is presumed to be 100,000. From this equation, it is apparent that for the same magnification, image brightness of both the illumination field and secondary fluorescence increases with the numerical aperture of the objective, but also decreases with increasing magnification. The actual numerical aperture and magnification values for a particular objective may vary by as much as 5-10 percent from the nominal values. Brightness transmitted by an objective, as discussed above, is also dependent upon the internal construction parameters, such as the number of glass lens elements, internal reflections and glare, and lens coatings. Often, it is necessary to sacrifice higher optical correction of chromatic aberration found in apochromat objectives for the enhanced image brightness displayed by higher numerical aperture fluorite objectives with lesser correction.
Use as high a numerical aperture condenser as possible with transmitted light fluorescence to avoid light loss and the accompanying long exposure times. Also when using a substage mirror on older microscopes, purchase a mirror that has an aluminized surface instead of a silvered mirror (silver will reflect ultraviolet light poorly). Often, an oil immersion darkfield condenser may be substituted for standard brightfield condensers for transmitted fluorescence applications. When using immersion oil, apply a non-fluorescent oil between the top lens of the condenser and underside of the microscope slide and between the objective front lens and the coverslip. Oils of this type are readily available from a number of commercial manufacturers.
Photobleaching, or dye photolysis, rapidly degrades fluorescent probes used to stain specimens and should be minimized by exposing the preparation to the lowest illumination levels as is possible. This effect is caused primarily by the photodynamic interaction between the fluorochrome and oxygen, which involves promoting the dye molecule from the singlet ground state to the relatively long-lived triplet excited state by a process termed intersystem crossing. Once the chromophore has been elevated to an excited state, it becomes more chemically reactive and may then participate in irreversible chemical reactions including decomposition, polymerization, oxidation (primarily by singlet oxygen) or reaction with another molecule. Reaction with oxygen will usually cause bleaching of the chromophore, depending upon the intracellular singlet oxygen concentration and the proximity of the chromophore to other internal cellular components such as proteins, lipids, or small molecules. Calculations indicate that singlet oxygen can produce chromophore photolysis over a distance exceeding 500 angstroms.
To minimize the effects of photobleaching, fluorescence microscopy can be combined with other techniques that are non-destructive to the fluorochromes, such as differential interference contrast (DIC), Hoffman modulation contrast (HMC), transmitted darkfield illumination, and phase contrast. The idea is to locate the specific area of interest in a specimen using the non-destructive contrast enhancing technique then, without relocating the specimen, switch the microscope to fluorescence mode. The results of a typical experiment of this type are illustrated in Figures 3 and 4. Figure 3(a) illustrates 3T3 fibroblasts in monolayer tissue culture imaged using phase contrast optics. The cell line was established from a National Institutes of Health line of Swiss mouse embryo cells, which are highly contact inhibited and useful for studies involving sarcoma virus formation and leukemia virus propagation. The photomicrograph in Figure 3(b) shows the same viewfield, but this time imaged using fluorescence illumination (a mercury vapor lamp and an Olympus WU filter cube) with cells stained by the fluorochrome 4',6-diamidino-2-phenylindole (DAPI), a nucleic acid specific dye with an emission maximum at 461 nanometers, which is used to selectively stain nuclei and chromatin. Figure 3(c) illustrates the two techniques used in combination to produce a beautiful photomicrograph of fluorescent-stained 3T3 cellular nuclei superimposed on a phase contrast image of the fibroblast cell membranes and internal organelles. This image was recorded with a specialized fluorite objective designed with phase rings to permit simultaneous observation of both fluorescence and phase contrast with the same objective.
Another example of combined techniques, this time using differential interference contrast (DIC), is presented in Figure 4. Illustrated in Figure 4(a) is a thin section of cat brain tissue infected with cryptococcus and imaged using DIC optics and a full-wave retardation plate. Note the pseudo three-dimensional appearance of the photomicrograph. Figure 4(b) shows the same viewfield, but imaged with fluorescence illumination and an Olympus WIB filter cube. The cells in Figure 4(b) were stained with a combination of fluorescein-5-isothiocyanate (FITC) and Congo red (emission wavelength maxima of 520 and 614 nanometers, respectively). The two techniques are used in combination to produce Figure 4(c), which illustrates the infected cat brain tissue in both fluorescence and DIC illumination.
Fluorescence Combination Techniques
Explore how contrast enhancing techniques can be coupled to fluorescence microscopy to locate specific areas on the specimen for further analysis.
The rate of photobleaching is dependent upon several factors, including the chemical reactivity of the chromophore, the intracellular chemical environment, and the intensity and wavelength of the excitation light. Some fluorescent dyes are readily susceptible to photobleaching while others are relatively insensitive and are stable for much longer periods. In many instances, the specimen may recover from the effects of photobleaching, particularly if it is kept cool and in a dark environment. Photobleaching should be distinguished from another fluorescence artifact termed quenching, which occurs by reduction (or in some cases, enhancement) of fluorescence intensity by competing processes such as temperature, high oxygen concentrations, and molecular aggregation in the presence of salts or halogen compounds. Sometimes quenching results from the transfer of energy to other acceptor molecules residing physically close to the excited fluorochromes, a phenomenon known as resonance energy transfer. This particular phenomenon has become the basis for a newer technique of measuring distances far below the lateral resolution of the light microscope. Impurities in the chromophore may also reduce fluorescence intensity either by photobleaching or quenching.
Photobleaching can be minimized by reducing exposure times (as discussed above) or by lowering the excitation energy (lamp intensity), however these remedies are accompanied by the undesirable effect of reducing the chromophore fluorescence emission energy. Neutral density filters can be placed in the light path before the light reaches the excitation filter, thus diminishing the excitation light intensity and reducing fading effects during specimen observation. Many fluorescence microscopes are equipped with a shutter system coupled to neutral density filters that allow excitation light to be reduced in intensity during observation and specimen manipulation, but enable the microscopist to easily switch to full-power illumination for photomicrography. It is also helpful, where possible, to deoxygenate specimens (but not living cells or tissues) and to use specific antifade reagents such as n-propylgallate and other inhibitors, which are commercially available. Chemicals that are capable of quenching singlet oxygen can also be employed to reduce the effects of photobleaching. Examples are 2-mercaptoethylamine, 1,4-diazabicyclo-2,2,2-octane (DABCO), diphenylisobenzofuran, p-phenylenediamine, and the amino acid histidine. Fading effects can also be reduced, in some instances, by changing the pH concentration of the mounting medium.
Figure 5(a) illustrates inhibition of the rate of fluorescence fading by several commercially available antifade reagents. The blue curve shows the rapid decline in fluorescence intensity with an untreated specimen, while the green and red curves demonstrate reduction of the fading rate by two antifade reagents of slightly differing efficiency. The specimen is marsupial kidney tissue culture cells (PtK2) stained with either fluorescein-5-isothiocyanate (FITC) or rhodamine phalloidin (both dyes have similar fading properties). Antifade reagents were p-phenylenediamine (green curve) or n-propyl gallate (red curve). Specimen secondary fluorescence fading rates usually differ depending upon the fluorochrome in use, even with observation under identical conditions. The best results can be obtained by selecting a fluorochrome with the lowest fading speed from those available for a particular application. This concept is illustrated in Figure 5(b), which compares the difference in marsupial kidney cell fading speed for three fluorochromes: rhodamine (yellow curve), the cyanine dye Cy3 (red curve), and FITC (green curve). Rhodamine, a ubiquitous dye with many applications, displays a very slow fading rate (Figure 5(b); yellow curve) under these conditions and is the best fluorochrome for photomicrography using this system.
To further minimize fading, it is advisable to conduct initial observations in one field within the specimen and then to quickly move to a "fresh" field just prior to taking the exposure. This practice may help to circumvent bleaching and/or fading effects. It is also helpful to do both observation and photomicrography in a partially darkened room environment. Although chemical methods and careful microscopy can reduce photobleaching, the most effective remedy is to increase detection sensitivity (coupled with reduced excitation energy) using a low-light level CCD digital camera designed specifically for fluorescence microscopy.
The occurrence of photobleaching has led to a technique known as FRAP, which is an acronym for Fluorescence Recovery After Photobleaching. This technique is based upon bleaching by short laser bursts and subsequent observation of the recovery of fluorescence caused by the diffusion of fluorochromes into the bleached area.
Barrier filters are designed to absorb specific wavelengths of excitation radiation that are reflected from, or transmitted by, the specimen and to transmit only the chosen visible light fluorescence. The cutoff wavelength range of the barrier filter is critical, because in many instances it must be able to transmit visible wavelengths that are not far in value from the excitation light used to illuminate the specimen. Because film is very sensitive to ultraviolet and visible wavelengths in the 380-450 nanometer region, it is particularly important that the barrier filter block all unwanted wavelengths in this range. To ensure removal of shorter wavelengths, additional ultraviolet blocking filters, such as the Kodak Wratten filter numbers 2A, 2B or 2E, should be added to the optical pathway. All of these filters completely absorb ultraviolet light, but have a slight difference in their absorption of lower wavelength visible blue light. Supplementary ultraviolet filters should be placed before the main barrier filter in the light path in order to block radiation that might induce autofluorescence in the main barrier filter, especially if this filter is yellow, orange, or red in color. If the barrier filter cuts the spectrum at too long a wavelength, specimen fluorescence color will be affected and the intensity of the image may be compromised, especially when only the long-wavelength portions of transmitted fluorescence are passed by the filter.
Color balance errors in fluorescence photomicrography may have a number of origins. The photomicrographs presented in Figure 6 illustrate several problems that arise with color degradation from autofluorescence and improper filtration when imaging specimens under fluorescence illumination. The specimen is a monolayer of fibroblast cells grown in tissue culture and stained with 7-amino-4-methylcoumarin-3-acetic acid (AMCA), a bright ultraviolet-excitable dye conjugate with an emission wavelength maximum of 445 nanometers (in the short-wavelength blue region). The optimal excitation wavelength of AMCA is 345 nanometers, which requires an excitation filter that passes a significant amount of ultraviolet light. Under proper conditions of illumination and filtration, the stained specimen appears with nuclei and cytoplasmic components stained a rich and deep blue color (imaged with an Olympus WU filter cube), as illustrated in Figure 6(b). When a barrier filter that passes too much ultraviolet light is used, a blue cast and background color are often present in photomicrographs of the specimen (shown in Figure 6(a)), and secondary fluorescence colors are degraded. Placing an auxiliary ultraviolet and/or short blue wavelength barrier filter somewhere in the microscope optical pathway is the method of choice for alleviating this problem.
Fluorochromes used to stain specimens often produce an undesirable amount of short wavelength blue autofluorescence, particularly when staining tissue sections. As mentioned above, this autofluorescence must be absorbed by the barrier filter to avoid degradation of secondary fluorescence emission produced by the fluorochrome-tagged specimen. Kodak Wratten filters 2A and 2E, which absorb short-wavelength blue radiation, are suitable for use in this application. The microscope manufacturers and aftermarket suppliers also offer filters useful in fine-tuning the cutoff range of barrier filters.
Intrinsic autofluorescence in mounting media can also cause the type of problems associated with the photomicrograph illustrated in Figure 6(a). Many of the popular permanent mounting media formulations exhibit autofluorescence as an undesirable side effect, and should not be used for quantitative fluorescence work. Instead, temporary mounts made with pure glycerin, glycerin-water mixtures, or fluorescence-free immersion oils should be utilized to prepare specimens. The microscope manufacturers offer specialized objectives that are optically corrected for specimen observation in glycerol. Autofluorescence produced by mounting media is usually pale blue or green in color and will lead to degradation of secondary fluorescence by the specimen. If the barrier filter does not remove superfluous autofluorescence generated by mounting media, sometimes Kodak Wratten color compensating filters can be added to the pathway to alleviate this problem. The pale blue color generated by most media can be removed by using a filter such as the CC20Y (yellow), and pale green fluorescence can be neutralized with a CC20M (magenta) filter. Use of higher density filters (CC30 and above) will often lead to absorption of secondary fluorescence.
Autofluorescence exhibited by cells and tissues in culture can often limit the ability to detect fluorescent probes in stained and fixed preparations. The extent of autofluorescence displayed by fixed tissues will often vary depending upon specimen preparation and the experimental conditions used to detect fluorescence. Flavin coenzymes (FAD and FMN) and reduced pyridine nucleotides (NADH) are the principal sources of autofluorescence in mammalian cells. Secondary fluorescence in fluorochrome-stained plant cells is often masked by autofluorescence from lignins (green autofluorescence) and porphyrins such as chlorophyll (red autofluorescence). This problem can sometimes be overcome with fixed cells and tissues by thorough washing with a 0.1 percent solution of sodium borohydride in phosphate-buffered saline for 30 minutes prior to staining.
When excitation and/or emission filters are not matched to the wavelength absorption and emission spectrum of chromophores used to stain specimens, then secondary fluorescence is usually compromised, as illustrated in Figure 6(c). In this example, an improper emission filter having too short of a wavelength cutoff is used to pass secondary fluorescence emitted by the specimen. Because a majority of this fluorescence is centered in the blue region of the spectrum (at 455 nanometers), only muddy reddish long wavelength color tones are passed by the filter. To correct the problem, a new barrier filter should be chosen. Overexposure of color film, along with reciprocity errors, can also cause dramatic color shifts of secondary fluorescence in final images.
Contrast errors often arise in fluorescence microscopy when the microscope optical train is mis-configured or the wrong filter combinations are utilized. Several of these errors are presented in Figure 7, which illustrates poor contrast in secondary fluorescence images caused by improper excitation filter choice and fluorescent immersion oil. The specimen is a cross section of a Japanese maple leaf petiole undergoing autofluorescence and imaged with a 10x fluorite objective using an Olympus WBV filter cube. Under ideal conditions, the image presented in Figure 7(b) would be obtained. However, when the excitation filter has too great a bandwidth (allowing unwanted wavelengths to pass), the image loses contrast resulting in the photomicrograph illustrated in Figure 7(a). Choosing the correct filter combination requires knowledge of the spectral values of excitation and emission of the chromophore(s) used to stain the specimen. In this case, the excitation filter passes wavelengths up to 550 nanometers, interfering with green secondary fluorescence emitted by the specimen and severely compromising image contrast.
Controlling specimen contrast in fluorescence microscopy requires that all optical components of the microscope be free of autofluorescence, and that they should not scatter light to a significant degree. This includes internal glass lens elements, mirrors, filters, and beamsplitters. Objectives should provide high image quality while transmitting light with high efficiency down into the near ultraviolet range. Filters must transmit desired wavelengths while blocking others that might interfere with observation of secondary fluorescence. Because excitation light is of far greater intensity than specimen fluorescence, the task of completely removing this light from the fluorescence image is not very easy and requires careful selection of filter combinations that provide a narrow range of excitation wavelengths (usually between 10 and 75 nanometers in bandwidth). When using high numerical aperture immersion objectives, it is critical that the immersion oil be free of contaminants that might exhibit fluorescence. Figure 7(c) illustrates an example of poor contrast generated by autofluorescence in immersion oil. Although the use of immersion oil helps to improve image brightness, partially by eliminating the loss of errant light caused by reflection from surfaces (in particular, the coverslip and glass lens elements), fluorescing contaminants that induce autofluorescence mix unwanted wavelengths with secondary fluorescence from the specimen. This has the unfortunate consequence of brightening the background while simultaneously reducing image contrast, both of which are undesirable.
Excitation and Barrier Filters
Explore color changes with excitation and barrier filter combinations in fluorescence microscopy.
Artifacts in specimen preparation will also produce contrast problems in fluorescence microscopy. Early pioneers in fluorescence often utilized non-specific fluorochromes that produced very bright images due to the fact that the dye molecules were randomly scattered throughout the specimen. Newer fluorescent techniques with improved and highly specific fluorochromes have resulted in specimens exhibiting stained target areas having substantially weaker fluorescence because much less dye is bonded. These newer methods require more careful specimen preparation to avoid unwanted fluorescence artifacts. It is often helpful to pre-stain portions of the specimen with non-fluorescing dyes that compete for binding sites to aid in fluorochrome selectivity. Specimens should be washed thoroughly to remove unbound fluorochrome before placement on acid-cleaned microscope slides made with non-fluorescent glass. Optimization of the fluorochrome environment by addition of chemical additives to minimize photobleaching helps to reduce or eliminate specimen fading. In addition, careful selection of the mounting medium chemical composition and pH is important to obtain the best image from secondary fluorescence. Coverslips and immersion oil should also be non-fluorescent, as discussed above.
Autofluorescence and/or non-specific fluorescence can occur as a result of the inherent properties of the specimen or through errors in staining. Typical errors of this type are illustrated in Figure 8(a), which shows a thin section of rat colon tissue stained with a mixture of fluorescein-5-isothiocyanate (FITC) and 4',6-diamidino-2-phenylindole (DAPI). The overall blue cast in the photomicrograph is due to a combination of autofluorescence and non-specific fluorescence due to overstaining with the DAPI fluorochrome. Proper staining with both dyes, followed by thorough washing of the specimen results in the photomicrograph presented in Figure 8(b). Errors similar to that illustrated in Figure 8(a) are also caused by using too broad an excitation bandwidth, a reversible mistake that can be corrected by use of filters with narrower bandwidths. Alternatively, a combination of excitation filters can be employed to selectively produce a specific set of wavelengths, or barrier filters can be adjusted to pass only longer wavelengths. Figure 8(c) illustrates the rat colon thin section when stained properly but now with a light red background caused by lack of a red suppression filter in the optical pathway. This error can be corrected by placing the proper suppression filter in the optical path.
Fluorescence photomicrography also suffers from common problems encountered with other forms of optical microscopy and photomicrography. These include focus errors caused by improper adjustment of the focal distance between the optics and film plane, microscope stand vibration, contamination by dirt and debris, underexposure, overexposure, color balance errors, specular reflections (common with long exposures and overhead room lighting), vignetting, and optical microscope mis-alignment. Examples of these errors are presented in our section entitled Fluorescence Photomicrography Errors with Color Transparency Film in the Photomicrography section of the Olympus Microscopy Resource Center Microscopy Primer.
Cameras and Film for Fluorescence Photomicrography - Improvements in film technology have produced increased film speeds and resolution improvements, but there are no color films currently available uniquely suited to fluorescence photomicrography. The specific properties of individual color films, including the characteristic curves for individual emulsion layers, properties of coupled dyes, and the spectral response to light used for fluorescence excitation and emission often vary dramatically from film to film and are sometimes difficult to control and reproduce. These problems lead to difficulty in maintaining accurate color rendering and exposure with color photomicrography using film.
Exposure measurement in fluorescence photomicrography is complicated by the fact that the brightness range within a fluorescent specimen is usually much greater than can be accurately recorded on film. Overexposure of the highlights will result in loss of fine detail and color saturation, while underexposure produces very dark images that do not reveal features hidden in shadowed areas. The background, which is usually very dark or black, is not as critical to either over- or underexposure. Three primary methods are utilized to determine exposure when using fluorescence: brightness measurement with a CCD, photodiode, or photomultiplier, continuous measurement during exposure (photomultiplier), and the least desirable method, trial and error. Modern photomicrography systems designed for fluorescence are equipped with the ability to gauge illumination intensity and calculate exposure times based on this reading. For routine fluorescence photomicrography, some cameras have a silicon blue detector cell for determining exposure duration, while others have a photomultiplier metering detector for measuring extremely faint specimens.
Film Properties
ISO Number | Light Sensitivity | Resolution | Grain Size | Exposure Tolerance |
25-50 | Low | High | Fine | Wide |
100-200 | Medium | Medium | Medium | Medium |
400+ | High | Low | Coarse | Low |
Table 2
It is important to consider several factors in choosing a detector system for fluorescence photomicrography. These include the detector's noise level and the signal-to-noise ratio for the image, spatial and temporal resolution, geometric distortion, linearity, and spectral sensitivity. In determining proper exposure for fluorescing specimens, it is best to use a camera with a CCD or photomultiplier metering system capable of accurate low-light measurement. Spot metering capability is valuable for measuring the area actually fluorescing and not the unwanted metering of the usual dark background surround the fluorescing material. Another technique involves the use of the exposure adjustment control to reduce exposure times that would otherwise be unduly lengthened by measuring the dark background of the field of view. In this respect, Olympus and Nikon offer camera systems that have built-in algorithms for reducing exposure if the camera is set for fluorescence mode. When light is scant (usually most of the time with fluorescence), set the trinocular prism to send all the light up to the film plane to reduce exposure time.
Ideally, a spot meter should be included in the photomicrography system to aid in determining exposure when only a few bright objects are visible on an otherwise dark background. For integrated light measurements made over the entire viewfield without a spot meter, the exposure time should be cut in half or to a fourth of the metered reading (for scattered fluorescing objects on a dark background), much like the situation with darkfield microscopy. If the spot meter light sensor is in a fixed position in the middle of the viewfield, the specimen must first be moved to the center of the field to obtain a suitable area for exposure measurement, then relocated to compose the photomicrograph. High-end camera systems allow translation of the spot meter sensor to enable the microscopist to determine photomicrograph composition prior to exposure measurement. After positioning of the specimen for photomicrography, the spot meter sensor can then be moved to the brightest area in the viewfield for exposure measurement without disturbing the specimen. When using a spot meter, make certain the fluorescent object is large enough to fill the spot and record an accurate exposure reading.
Some photomicrography camera systems provide a permanent beamsplitter assembly that directs a portion of the light to a photometer, which allows light measurement during the actual exposure in order to compensate for fading. This is a convenient method for adjusting exposure times on the fly to avoid photobleaching artifacts, but it has the disadvantage of reducing light intensity directed to the film, necessitating a longer exposure. More advanced computer-aided camera systems are able to compensate for reciprocity failure and fading through a pre-programmed lookup table conveniently stored in the metering system's memory. Automatic correction for reciprocity failure by camera system computers operate by applying the following equation to exposure calculations:
where Tc is the corrected exposure time, Tm is the metered (uncorrected) time, and p is a constant determined from reciprocity values for individual films housed in the system's lookup table. This system is limited by the amount and type of reciprocity data for films that are added to the lookup table during initial programming of the metering system.
Without an integral exposure metering system, determination of exposure time must be made by trail and error. This method involves making a series of trial exposures at a variety of exposure times, stepped through increments of one-half to a full f-stop (bracketing). After processing the film, the exposure that produces the best result is carefully noted and provides the basis for further photomicrography using the identical microscope configuration. Changes to specimen illumination or microscope setup will necessitate another series of exposure brackets to determine the new optimum exposure time. If only the objective magnification is changed, new exposure times can be calculated from previously recorded bracket data. Many photomicrographers doing fluorescence microscopy bracket their exposures, both over and under by three or more intervals, to make certain that they will secure a correct exposure at one of the settings. Keeping good written records of exposure and associated equipment data can reduce or eliminate the need for future bracketing. When determining exposure using the trial and error method, it is wise to continuously monitor the microscope lamp output for deviations in illumination that occur as the lamp ages. Microscopists using this method of exposure determination often rely on SLR cameras to capture images with fluorescence illumination. With these cameras, movement of the shutter and viewfinder mirror during exposure may cause vibrations that lead to unsharp images. This problem can usually be circumvented by using a remote shutter switch and/or raising the mirror prior making the exposure. In some cameras, operation of the self-timer releases the mirror, which allows time for vibrations to cease before the exposure is made.
Illuminated Reticles
Practice examining fluorescence specimens with illuminated reticles by adjusting the focus and color of the reticle.
Microscopes equipped specifically for fluorescence illumination often have an illuminated reticle in the system or focusing telescope, which allows the microscopist to visualize a photomicrography grating superimposed over faintly visible specimens positioned against a very dark background. Both Olympus and Nikon provide reticles in their high-end microscopes that offer a choice of crosshair and film frame colors with which to view the specimen. Microscopists can choose between a red or yellow illuminated reticle. The red reticle has the advantage of maintaining the dark adaptation of the observer's eye and provides excellent contrast when viewed over blue and green secondary fluorescence. For red fluorescence and when simultaneous phase contrast or DIC are used, the yellow reticle should be selected to maximize contrast. Some manufacturers offer a focusing telescope with illuminated framing reticle that attaches to the automatic camera housing and allows viewing of the film frame dimensions over the dark background field.
When choosing film for fluorescence photomicrography, the film speed (ASA or ISO rating) is a variable that should be carefully considered. The higher the ISO rating, other things being equal, the less the amount of light necessary to register a satisfactory image on the film. Illumination sources used in fluorescence microscopy require daylight-balanced film emulsions to accurately render specimen secondary fluorescence colors. Most major film manufacturers offer a wide spectrum of daylight films at a variety of ISO ratings in both transparency and color negative formats. Kodak daylight Ektachrome with an ISO of 200 or 400 or Kodachrome 200, both in 35 millimeter size, are positive transparency films that display adequate speed, good color rendition and acceptable resolution. Fujichrome Provia is offered in 400 and 1600 ISO ratings, and is an excellent transparency film in terms of color saturation and resolution. Other manufacturers make comparable transparency films, but we suggest avoiding the use of color negative film if at all possible.
For black & white film, Kodak offers T-Max 400, an excellent fine to medium grain film that is one of the best for fluorescence applications. Several black & white films are available that can be processed with standard color negative chemicals (C41). These include Kodak T-Max 400CN, Kodak Advantix, and Ilford XP-2 Super, all of which are offered in ISO ratings of 400. Keep in mind that faster films allow for much shorter exposure times at the expense of increased grain size. Table 2 lists basic film properties for reference.
Contrast, one of the greatest concerns in fluorescence photomicrography, depends not only on microscope configuration, but also upon the physical characteristics of the film chosen, the development process, exposure details, and the specimen itself with respect to the efficiency of staining by the fluorochrome and photobleaching or fading. Color transparency films can be underexposed by one or several f-stops (a bonus when considering reciprocity and fading), then push processed by extending processing time in the first developer to increase contrast and color saturation. Many black & white films can produce variations in contrast that are dependent upon the developer mixing conditions and processing time. Resolution of film in fluorescence photomicrography is determined principally by the optical parameters of the microscope (primarily the objective numerical aperture) and how well it is configured, but is also somewhat dependent upon film characteristics and development methods. In practice however, the film resolution only becomes a factor when the microscope is not performing optimally or the ISO speed is so high that grain becomes a factor.
Recent advances in digital imaging technology have revolutionized capture of photomicrographs using charge-coupled devices (CCDs). When imaging multiply-stained fluorescence specimens, digital cameras provide an excellent solution and are rapidly becoming the medium of choice for many photomicrographers. The price of three-chip integrating color CCD cameras has dropped to an affordable range and, when coupled to a frame-grabber or digital video card and computer, offer an excellent method of collecting, digitizing, editing, and storing fluorescence images.
The weak fluorescence displayed by many specimens necessitates high performance in CCD detector specifications. Currently, the best choice is a back-thinned frame or interline transfer device, which is equipped with heat sinks and/or external reservoirs for cooling. Many of these cameras have a quantum efficiency approaching or exceeding 90 percent and permit the sequential acquisition of several images before readout. Image acquisition cycles run from one to five seconds, depending on the readout rate, video capture parameters, and CCD resolution. A typical CCD camera matching these specifications is the Optronics MagnaFire digital imaging camera system designed for scientific applications. This camera has a SONY ICX085AL 2/3-inch interline transfer CCD with an array size of 1300 × 1030 and a (square) pixel size of 6.7 microns. Camera performance is enhanced with a 60 dB dynamic range and signal-to-noise ratio, a file bit depth of 24, 32, or 48 bit color, a dark current of 4 electrons per pixel/second with a 16,000 electron well depth, and sealed gas Peltier CCD cooling. Color image formation is via multiple dichroic optical glass color filters. Optronics and other manufacturers also offer a wide spectrum of digital cameras (a typical CCD camera is illustrated in Figure 9) suited to almost every need in both fluorescence and routine optical microscopy.
The following points summarize important aspects of fluorescence photomicrography.
- Use high numerical aperture objectives equipped with glass or quartz lenses that are transparent to near ultraviolet light. Also use the lowest magnification projection lens possible to limit the total magnification.
- Reflected light fluorescence is preferred to transmitted light fluorescence. If it is not possible to configure the microscope for reflected light, use a transmitted light darkfield condenser.
- Optimize specimen contrast by careful choice of film parameters, proper microscope configuration, and by carefully matching chromophore excitation and secondary fluorescence properties to the proper excitation and barrier filters.
- Remove the trinocular beamsplitter prism from the light path to allow 100 percent of the light to reach the film plane during exposure. Also use 35 millimeter film and avoid larger film formats.
- Choose daylight balanced transparency films for the best rendition of specimen colors, resolution, and contrast. Push process transparency films for enhanced contrast and color saturation.
- The light source must provide high-intensity radiation in very narrow ranges of the spectrum, typically between 10 and 50 nanometers. The best choices are xenon and mercury vapor lamps or lasers tuned to specific wavelengths.
- The microscope optical train, including lenses, mirrors, filters, beamsplitters, immersion media, microscope slides, and coverslips, should be free of autofluorescing components.
- When not viewing or photographing fluorescence specimens, block the excitation light using the filter slider or shutter in the illuminator to avoid specimen damage by photobleaching.
- Always provide a heat filter between the illuminator and fluorescence filters, which may be damaged by excess heat.
- Monitor the lamp for signs of flicker and replace when illumination intensity becomes uneven. Keep the lamp and vertical illuminator adjusted for proper Köhler illumination.
- Prepare specimens carefully and remove excess chromophores by thorough washing after staining.
In conclusion, the field of fluorescence microscopy is rapidly expanding in many medical and biological research laboratories. The mainstream of fluorescence microscopy has undergone an almost total shift from utilizing transmitted light to incident light, accompanied by the introduction of many new and different fluorochromes. This has been an impetus for spurring many new and varied fluorescence microscopy applications.


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Balornock Primary pupils
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I take English Lit 12 through SIDES, our local distance education school. It is a very exciting course…I had been looking forward to taking Lit 12 at GNS with one of my favorite teachers since grade 9. He read for us passages from Chaucers' "Canterbury Tales" in the traditional old English, and "Beowulf" in the Anglo-Saxon tongue. I was inspired and enthralled that something someone hundreds of years ago had spoken and written had survived, and that I could still connect to the characters, and laugh at the follies of the pilgrimages and their fascinating glimpse of 14th century life, and cry on the beach as Beowulf lay, dying, but triumphant against his last fire-breathing foe. I had been hooked on reading ever since I got past the first big hurdle of actually learning to read. I was against the idea, determined to never read, but instead work at a gas station or McDonalds. Times have changed.
I am currently working my way through the "Canterbury Tales", which I spent the greater part of today working on. I have a wonderful audio-book version of the poem, which saves me the headache of wading through the soup of old-English's abundance of odd vowels and consonants, whose sound has evolved over the years.
To complete a 'literary-ily' perfect day, I scored big points with the rare word qiviut, coming to us from the Inuktitut word ᕿᕕᐅᖅ. I feel like such a fibre nerd. I am slowly picking up drops of energy, but am trying to rest up for whatever excitements of life await me this week!
Qiviut: the wool of the musk-ox |


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Do you dress up with your kids?
I always do.
Do you pick out your spouse's costume for them?
Do you visit haunted houses?
I haven't in quite a while but they're fun!
I wanted to dress up this year, but I wanted to do the couple costumes. I wanted to be a pregnant nun and TNT be a priest. But with me working in an office and him selling tools I don't think people would get the whole picture if we weren't together.
My sis and her friend are going as a pair of testicles!
We typically don't dress up...maybe when the kids get older.
And, a big no on the haunted houses...I don't like being scared.
I don't dress up with my kids, but I would like to, so maybe one year....
I have visited a haunted house or two in my time, but not recently.
My daughter is quite creative with her costumes, we spend forever going around all the costume places and then she ends up designing her own! This year she is a Jedi and my youngest is going to be tigger. My oldest isn't dressing up this year
I never dress up for Halloween, but The Man does sometimes when he takes the girls trick or treating. Usually something homemade and last minute and fairly hilarious.
And that would be a giant NO to haunted houses. My heart can't take it.
I usually slap on a kitty ear headband and call that a costume.
No spouse... no picking.
Was SUPPOSED to go to a haunted house with Mr. B but... we just didn't get there.
I used to totally dress up, but the past few years it hasn't really been ideal, so I would slap on a hot pink bobbed wig with pink lepoard spotted ears attached to it, slather on a black spot on my nose and scribble some whiskers on my cheeks and call it good. I would also wear a halloween t and socks. I was soooo stylin'. Not sure if I will do anything tonight. I am soooo not feeling Halloween this year.
Yeah, I know. Party pooper. :p
Happy Halloween!!
I forgot....
No spouse...but if I had one, no picking. Let him figure it out. I have a hard enough time picking one for myself. Sheesh.
And haunted houses...eh, I like coming home with dry britches. Thanks for thinking of me, though. :)
I need to go back to being more fun and start dressing up. Next year! Those pics are great!!!!!
I will definitely be goig to haunted houses when I know Tristan can handle it. Happy Halloween!!!
Great costumes!
I usually don't dress up.
Light of my life always picks out her own costumes (except when she was real small).
This year, she's a fairy. I'll post pics when I have them.
Great photos of your family!!! I really enjoyed the peek inside casa de tangle.
I wore a funny sweatshirt...read my post to see it.
I picked out HWMs costume but he likes to be really creepy scary and that still bothers me.
We try to go to the a haunted hayride and to Busch Gardens Hallo-Scream events. I love this time of year.
OMGosh, y'all are too stinkin' cute!!! I mean, scary. No, adorable. No .... :-)
Happy Halloween to you all.
Did the yellow hat!!! Happy Candy Day!
My husband never dresses up! I occassionally do it just depends on the year and what mood I'm in! :)
Do you eat all the candy in two days?
I do.
Kit Kat
Reeses's Peanut Butter Cup
Sugar Stix
Put them in my bag, please.
I have never dressed up with the kids. And today I had lunch with Madalyn at school...there were lots of moms there in costume. I had no idea...
Michael doesn't dress up.
And I don't do haunted houses. I don't like to be scared...
Cool costumes!!!!
My favourite is Capt Sparrow...but then I'm a bit biased in that direction... :-)
I dressed up last year .. as a Witch.
I'd be picking out J's costume if he was going to be wearing one..
yep, I love haunted houses. Didn't go his year though. Halloween sort of sneaked up on me..
I haven't dressed up for Halloween since I was in the 6th grade... I get a Halloween tee from Old Navy every year.
Shane does not dress up, either.
And I don't do haunted houses. For me, Halloween is all about the candy. Don't wanna be scared!
remy is so little! cute!
im dressing up as bella cullen and i was going to make my boyfriend be edward, but that just seemed wrong (esp since he didn't read the book... he's a law student and as im sure you know, doesn't have time for fun reading) so he's wearing the "second amendment" shirt (if you havent seen it go here: http://fashionablygeek.com/wp-content/uploads/2008/05/the-second-amendment.jpg) so i sewed him bear arms to wear under the shirt :)
remy is so little! cute!
im dressing up as bella cullen and i was going to make my boyfriend be edward, but that just seemed wrong (esp since he didn't read the book... he's a law student and as im sure you know, doesn't have time for fun reading) so he's wearing the "second amendment" shirt (if you havent seen it go here: http://fashionablygeek.com/wp-content/uploads/2008/05/the-second-amendment.jpg) so i sewed him bear arms to wear under the shirt :)
Nope, my boyfriends doesn't dress up....Oh, well!!!! Theres always next year. GREAT costumes!!!
Happy H-Ween!!
- Jennifer
I love love love haunted houses!
have a great one!
It depends on the intensity of the haunted house. Usually no. I am a big wuss! I will dress up with my kids when I have some. :) We (my hubby and I) usually dress up if we have a party to go to. Fun stuff! I have never seen a jack sparrow before. Love the eyes!
I don't dress up on halloween anymore-- it seems like all my energy is gone by the time I have everyone dressed. What were you this year???
Happy Halloween!
I haven't costumed up in a long time. I'm thinking of having an adult costume party next year.
And yes I LOVE haunted houses!!!
next year, Jeff and I are so dressing up with the kids.
I want to dress up with the kids - and I REALLY want to do a dorky theme dress-up. You know, like the ketchup, mustard, and pickles or something. That would be totally hawt.
I sometimes dress up with the kids. One year, I was Dora's grandma (guess what she was:-).
I dressed up as an unemployed stay-at-home-dad. I think I nailed it.
Everyone looks great. Love that sombrero on your dad! I was going to dress up but things took a sour turn this morning and I lost my enthusiam. Bummer.
I haven't dressed for quite a while. I've gone through stages. Before kids I did dress up parties. During kids I had a blast creating their costumes, now after kids I am looking into doing the dress-up thing again. Fav costume I saw tonight was Edward Scissorshands.
Little one's double fisted with swords eh?
Rhea, these are SO cute!!!
Cute pictures! I don't have the energy (or extra $$) to dress up along with the kids. But we had a good time anyway!
Girl, you are so like me. I know what you are. You are a witch that later found a cute sparkly red mask you just had to wear. I'd do something like that!
I ALWAYS dress up. Maybe not always something super fancy, but always something. Halloween is my very favorite holiday. I was at a carnival last night and can't get over the amount of adults that do NOT dress up. Who can pass up being a kid?
I try to dress up, but this year I didn't make it...too busy trying to get the five girls all ready. I dressed the baby :) so that counts :)
I love Halloween because we didn't have it in Italy and I feel I need to catch up on all the years I didn't get candy....
I sometimes dress up with the kids. I dressed hubby up one year. Haunted houses scare me!
I haven't dressed up with my kids yet, but I'm going to next year. For sure! And my hubby would rather walk on glass than dress up. Not his style. I don't do haunted houses because my heart can't take it. I don't watch scary movies either. Yeah, I'm a wimp.