Objective lenses: There are usually 3-5 optical lens objectives on a compound microscope, each with different magnification levels – most commonly 4x, 10x, 40x, and 100x. The total magnification of a compound microscope is calculated by multiplying the objective lens magnification by the eyepiece magnification level. So, a compound microscope with a 10x eyepiece magnification looking through the 40x objective lens has a total magnification of 400x (10 x 40).

Please read on to Part III to learn a new photography term you didn’t know you needed. In Part IV, I will share a quote that will forever change how you approach setting your exposure.

In a standard bright field microscope, light travels from the source of illumination through the condenser, through the specimen, through the objective lens, and through the eyepiece to the eye of the observer. Light thus gets transmitted through the specimen and it appears against an illuminated background. The observer can see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are just thick enough (but not too thick) to absorb a significant amount of light despite being colorless (Caprette, 2012; New York Microscope Company, 2019).

Bright-field microscopy allows one to observe the development, organization, and function of unicellular and higher organisms and to study structures and mechanisms at cellular and subcellular levels (Periasamy, 2014). A common application of the upright compound light microscope is in cytogenetics i.e. the study of chromosomes. As an example, the steps and materials used to prepare and observe metaphase chromosome spreads of a marine mollusc is described below (based on Van der Merwe and Roodt-Wilding, 2008):

The design described here is for a standard upright compound light microscope. Another design commonly used, especially when observing living cells, is the inverted microscope. The difference is in where the objectives sit, where the light source is, and which parts of the microscope move to bring the image into focus. In the conventional upright microscope (as in Figure 1), the objectives are attached to a nosepiece on the microscope body above the stage, the sample is illuminated from below, and the focus controls move the stage up and down to bring the image to its proper location of focus relative to the eyepiece. In inverted designs, the stage itself is fixed and the objectives are below the stage, in an inverted position. The sample is illuminated from above and the focus controls move the objectives up and down to focus the image in the eyepiece. Having a fixed stage allows better access to the specimen in circumstances where the specimen needs to be manipulated while being observed (such as microinjection) (Murphy and Davidson, 2012).

Higher ISO settings amplify the signal more, making the sensor appear more sensitive to light. This allows you to capture images in darker environments without needing longer exposure times or wider apertures.

The last sentence may have caught you a little off-guard. Let’s review a few basics, and then I will explain in detail why ISO is not an exposure setting in a digital camera.

Bright field microscopyand darkfield microscopy

Many argue that ISO has never been a part of exposure, even for film. I definitely believe that ISO is not and never has been an “exposure setting,” but it certainly impacts your other exposure settings.

At the base ISO setting, the signal is amplified to a certain level. Increasing the ISO setting further amplifies the signal, essentially “boosting” its strength and making the image brighter.

While all this development was occurring, engineers, likely with some input from marketing people, realized that to get photographers to adopt this new revolutionary technology, they would need to make the transition as smooth and straightforward as possible.

While some amplification happens at the hardware level, most modern cameras also apply software-based “gain” adjustments after capturing the raw data. This helps mitigate noise but also introduces its own subtle processing artifacts.

Light (or Illumination): The light used to illuminate the specimen from the base of the microscope. Low voltage halogen bulbs or LED are the most commonly used sources of illumination for compound microscopes.

Bright field microscopyprinciple

Specimens are often stained to enhance contrast. When this applies, the specimen or tissue can either be stained before it is mounted on the slide, or the stain can be added to the mounting medium.

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Unfortunately, amplifying the signal also amplifies existing noise within the sensor itself. This manifests as unwanted grain or speckles in your images, particularly at high ISO settings.

ISO, even though it is a gain setting, was still referred to as adjusting the sensitivity of the camera’s sensor, and the big lie began.

Stage adjustment: Adjusts the position of the mechanical stage horizontally in the X and Y plane, in order to position the slide so that a portion of the specimen is under the objective.

Microscopy is used to visualize objects that are too small to see with the naked eye. In biology, this technique enables us to examine things like bacteria and cells at a magnification of up to 10000 times their original size (BIO1000F practical manual, 2017). In light microscopy, visible light is used to detect such small objects – with bright field microscopy being the most common form of light microscopy. In bright field microscopy, the image you see is formed mainly by the absorption of light by the specimen (for example a cell) (Lackie, 2010).

Bright field microscopyadvantages and disadvantages

Yes, you have to “set” the ISO on your film camera, but that is just to inform the camera’s light meter what speed film you have loaded. It is not a setting that you choose on a frame-by-frame basis.

In 1991, Kodak created the first-ever digital SLR. The Kodak Digital Camera System (DCS) was a modified Nikon F3 whose film chamber and winder were modified to make room for digital sensors. The camera had a built-in 1.3-megapixel Kodak CCD to capture images.

This article is the second of a four-part series on exposure and the fact that our industry has yet to keep up with technological changes regarding how we think about, talk about, and teach exposure.

Nosepiece: Holds the objective lenses and attaches them to the microscope head. The nosepiece rotates to change which objective lens is in the working position.

Darkfieldmicroscope

In 1975, Steven Sasson, an engineer at Kodak, developed a portable, battery-operated, self-contained digital camera. It weighed 8 pounds (3.6 kg) and used a Fairchild CCD image sensor having only 100 × 100 pixels (0.01 megapixels). The black and white images were digitally recorded onto a cassette tape, a process that took 23 seconds per image.

I hope you found this information useful. Now go pick up that camera and shoot something! Because – “Your BEST shot is your NEXT shot!” — Joe Edelman

Cytogenetics is just one of many applications of the microscope in the biology laboratory. Whether it is used for identifying cells and tiny organisms, or to study the organisation and processes within cells through live-cell imaging, there is no doubt that bright field microscopy has advanced the life sciences over the past decades. With increasing sophistication and technology, this technique promises to stay central to the study of life.

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That equation is how exposure has been considered since the beginning of photography in the early 1800s. But that equation has been inaccurate for digital photographers for over 20 years.

To understand this, we need to know that the sensors in our digital cameras are analog. (You read that correctly – analog, not digital)

Together, these two components form the core light-collecting and signal-generating unit of each pixel in a digital camera sensor. They work in tandem to convert light into an electrical signal.

Each pixel on a digital camera sensor is like a tiny solar cell, converting light into an electrical signal proportional to the light intensity.

Fast forward to 2023, and the camera sensors, firmware, and software are much better at interpreting the higher ISO sensitivities. We also have incredibly effective noise reduction software, which is being improved dramatically by AI.

Much like politics, we may never get people to agree on one definition. Still, regardless of which side of the photography aisle you are on – there is only one truth because the truth is based on physics, electronics, and engineering.

I realize it is hard to imagine photographers complaining or whining about anything (Think AI, Competition, Customers in general… Wait, that is a whole different article).

Aperture refers to the size of the opening within the lens through which light passes to reach the sensor. A wider aperture (lower f-number) allows more light, making it ideal for low-light situations. Conversely, a smaller aperture (higher f-number) lets in less light, which is useful for bright scenes or achieving more depth of field.

There are creative options where you would choose slower shutter speeds, such as intentional motion blur (ICM), which can convey a sense of movement or add a dreamy, ethereal quality to the image.

Every roll of film has a set ISO or sensitivity based on its chemical makeup and the chemical reactions that occur when the film is developed.

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Bright fieldmicroscope application

As a holdover from the film days, we have been told that raising your camera’s ISO increases the camera’s sensitivity to light. Nothing could be further from the truth.

Digital camera sensors do not record light the same way that film does. Everyone acknowledges that; however, as an industry, we can’t get everyone on the same page about how to talk about how digital camera sensors work and how exposure is managed with them.

It is time to realize that there are more accessible and better ways to teach this technology, beginning with the updated definition of ISO I just shared with you.

Eyepiece (or ocular): The part that you looked through at the top of the compound microscope. Eyepieces typically have ocular lenses with magnification between 5x and 30x. Many microscopes are fitted with foldable rubber eye guards that can help minimize ambient light.

Condenser: Condenses the light from the base illumination and focuses it onto the stage. The condenser has an iris diaphragm (a circular opening where light can pass through), which can be adjusted to match the effective numerical aperture of each objective lens. To open and close the iris diaphragm, the condenser ring can be rotated so that the amount of light permitted through matches the requirements of the objective in use.

Smaller apertures with their extreme depth of field help ensure that everything in the scene stays in focus. Wider apertures can be used creatively to draw attention to your subject. Depending on your subject matter, both options dramatically impact the feeling or mood of the photo.

This amplification increases the perceived sensitivity to light, allowing you to capture images in lower lighting conditions.

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Fine and coarse adjustment controls: Adjust the focus of the microscope by moving the stage vertically. The coarse adjustment knob is moved to its highest position stop (forward rotation). The fine adjustment knob is used to bring the image into sharp focus.

The typical upright compound microscope consists of the following parts (see figure 1, from the bottom up) (BIO1000F practical manual, 2017; New York Microscope Company, 2019):

Figure 1. Labeled parts of a microscope (Adapted from Thebiologyprimer, 2014 Wikimedia Commons / Public Domain https://commons.wikimedia.org/wiki/File:Parts_of_a_Microscope_(english).png)

My intent with this series is to educate you about what we are overlooking and offer solutions that you can use to improve your photography immediately.

With the inverted microscope, living cells can be observed in their culture dishes with medium on the microscope stage. This is called live-cell imaging and it enables the observer to monitor a variety of dynamic intracellular events over time. Because the inverted microscope has a fixed stage onto which culture dishes or flasks can be viewed, no special specimen preparation is necessary. Live-cell imaging often makes use of fluorescent labeling to help the observer distinguish cellular structures and processes (Murphy and Davidson, 2012).

It is time to catch up to make the most effective use of the gear we spend thousands and thousands of dollars on. It is time to take advantage of the fact that our cameras can do much more for us than film cameras.

If we find an easier way to discuss it, we will make it easier for more people to understand. Let’s set the record straight.

The coordinated system of lenses is arranged in such a way that a magnified image of a specimen can be viewed with increased resolution and contrast. Resolution is how well one can distinguish between two points on a specimen – the better the resolution, the sharper the image. Resolution is a function of the microscope, its lens design and source of radiation. Contrast, on the other hand, is the difference in intensity perceived between different parts of an image. Histological stains that bind selectively to different parts of the specimen can enhance the contrast of an image (BIO1000F practical manual, 2017; Lackie, 2010). Contrast is thus mainly dependent on intrinsic qualities of the specimen and how it was prepared for microscopy.

Bright fieldmicroscope image

Darkfield microscopy

The work done by the camera’s processor and firmware interprets what we historically knew as ISO or sensitivity. Please note the word “interprets”. The ISO or sensitivity is not changed; it is interpreted.

Fast forward to our digital cameras, and while we still have an ISO setting, the fact is that ISO is applied after the exposure has been made.

Light intensity control: Knob is used to adjust the amount of light that reaches the specimen or slide from the base illumination.

Advantages ofbright-fieldmicroscope

Depth of field is the range of focus in your image. The distance in front of your focal point to the distance beyond your focal point that appears to be in focus.

The camera’s processor then converts the amplified signal from analog to digital (A/D). The digital data is further processed by the camera’s firmware, correcting for color variations, white balance, and other factors.

Because light needs to travel through it, the material you observe with the compound microscope must be very small, transparent, or cut in a thin section. Preparing specimens for viewing under the upright compound microscope involves placing them on a glass slide in a mounting medium, such as water or glycerine. A coverslip is then placed over the specimen to protect the lens from the mounting medium and to flatten the specimen slightly. Throughout the wet mount specimen preparation, care should be taken to minimize obstructions (like debris or bubbles) in the light path. The idea is to create as clear as possible, a path for the light to travel through. Here follows a protocol for preparing a wet mount:

Shutter Speed is the camera setting that controls how long light is exposed to the sensor. Longer shutter speeds (seconds or even minutes) let in more light, useful for low-light situations like night photography. Conversely, shorter speeds (fractions of a second) let in less light, which is ideal for freezing fast-moving subjects or capturing bright scenes without overexposure.

We are more than two decades into the digital era and already in the second generation of digital cameras (mirrorless), and the way we talk about exposure is still the same way we talked about it in the last century when everyone shot film.

Ironically, even in 2024, Nikon’s website on one page refers to ISO as signal gain, and on another, states, “increase in ISO will make the camera’s sensor more sensitive to the lack of light.” Yes, even their marketing and education people don’t have a solid handle on it.

The simple solution for Kodak and Nikon was to keep the same settings that photographers were comfortable with. For other manufacturers, it was a matter of why rock the boat; it just made sense to follow along.

Photographers who adopted digital cameras in the early 2000s probably still suffer from a form of digital noise PTSD because raising your ISO two to three stops above the base would render an image almost unusable.

Different sensor types vary in their inherent sensitivity and noise characteristics. Full-frame sensors tend to be better at low light performance than smaller sensors, allowing them to offer higher usable ISO ranges. This happens because full-frame sensors have more real estate to use bigger photodiodes.

Light microscopy has become one of the most widely used methods in the life sciences since the invention of the microscope in the 1670s by Antoni van Leeuwenhoek. Soon after the first microscope was built, a host of biological specimens (including protozoa, bacteria, spermatozoa, and red blood) were observed and described (Periasamy, 2014). Nowadays, no biological laboratory is complete without a microscope

A simple microscope is really nothing more than a magnifying glass, where a convex lens is used to magnify an image. A compound microscope, on the other hand, uses a minimum of two magnifying lenses or lens arrays, called the objective and the eyepiece (BIO1000F practical manual, 2017; Encyclopædia Britannica, 2019).

Shutter speed is also useful when hand-holding a camera to ensure that you will not introduce camera motion because of shaky hands due to heavy gear. This type of motion blur is often confused for an image that is soft (slightly out of focus – but not intentionally)