Basically, a typical microscope is basically a box designed for storagetwo lensesin precise positions so that the light can be precisely magnifieda sampletowardsdetector. The first of these two lenses islens,which is near the sample moves whenfocus knobis rotated and contains useful information such asincreasewritten on your side. The latter is commonly known ascollecting tube/lens, which is hidden deep in the body of the microscope and is rarely seen.
In addition to these lenses, microscopes consist of:
- light sourceslike a lamp or a laser
- Detector, usually a scientific apparatus
- lens, a binocular-like device that allows the user to observe the sample directly
- Sceneryfor the sample to settle or mount
- mechanical controlsuch as apertures, filters, dials to manipulate the light path or the position of the lenses
- digital controllike microscope software where you can control factors such as exposure or field of view
- Extra lenses/mirrorsto further manipulate the path of light
These components are responsible forincrease,resolution,yfield of viewcharacteristic of the microscope. This article explains the details of the components and anatomy of the microscope in relation to how they contribute to providing the best possible image. For the location of these components, seeFigure 1.
Aspringthis isoptical devicethat can refract light. Refraction depends on the shape of the lens, which is usually the caseconvexoconcave. For microscopic purposes, convex lenses are used because of their abilitycenterlight at one point. This is how the human eye works: a convex biological lens focuses light at the back of the eye, where rods and cones can detect it. Microscopes borrowed this idea and used convex lenses to focus light to a specific pointFlens distance. This distance is calledfocalthe length of the lens and depends on the shape. Lens shapes can be seen inFigure 2. Note that these lenses are symmetrical and will have the same effect on light in both directions.
The focal length of the microscope objective lens must be very small because the objective lens is usually very close to the specimen. Typically, the higher the magnification, the closer the target should be.
Microscope lenses contain lenses, but they are not as simple as those seen inFigure 2do themcomplexglasses (Rysunek 3A). While the overall effect can be magnifying, these lenses have been carefully designed to control various aspects of the lens such asworking distanceand problem solving skills such asaberrations. Goals are characterized by two factors:increaseynumerical opening (NA). The magnification of the objective is from 2x to 100x (and is combined with the magnification of the eyepiece), magnifying the specimen respectively from 2 to 100 times (Figure 3B). NA is related to the focal length of the lens, i.e. at what angle the light enters/exits the lens, as this affects the resolution (Fig.3C, read our application note on resolutions and NA for more information). SeeDig. 3for more information.
The field stops and opens
There are always limits to the imaged area and the detailed information provided by the microscope. Isphysical blockson the path of light, usually calledstops,membranes, oholes. The term stop will be used here. For the image path, these may or may not be adjustable by the user, but as discussed below, the same concepts apply to lighting optics.
Onopening stopthis is part of the imaging system that limits the range of light angles the lens can collect from the sample. This range of angles defines the NA lens and hence the resolution of the system, the ability to detect two objects as different. Most microscope objectives are designed so that the aperture stop is in positionobjective posterior apertureas seen inRysunek 3A. This allows the lens to determine the resolution of the system and the resolution is the same throughout the field of view.
Onfield stoplimits the area that can be photographed. It cannot be larger than the diameter of the pipetube lens. In the best case, the area photographed is the diameter of the lens divided by the magnification. If the inner lens has a diameter25mmand magnification is100xa circle with a diameter of 250 µm should be visible from the sample. Elements such as light modifying elements or the detector itself can easily reduce the recorded field of view.
The output of most microscopes is an image about 2 cm wide, so this is usually the caseenlarged againfill the field of view of the eyes.glasses, another magnification system, offers magnification from 10x to 30x in addition to that provided by the objective and microscope. Combined with the eye lens, this magnifies the image on the retina to a useful scale, allowing the human eye to distinguish and observe objects as small as cells (~10 µm).
There are many types of cameras that can be used with a microscope. The key experimental considerations aresensitivity,resolution,field of viewyspeedcamera. A detailed explanation can be found in our articles on these topics.
Apixel apparatusit is the camera's individual light measurement unit, and the camera's sensor contains arrays of pixels to measure the light in the field of view. The camera can be from 128 × 128 pixels to 5000 × 3000 (15 million pixels or 15 megapixels) or more. Since the ports of microscope cameras are usually about the same size, cameras with larger pixel arrays often have smaller individual pixels.
Pixel sizeThe key is the ability to obtain images that contain all the information content provided by optics. Camera pixels are square and are typically 3 to 24 µm long along the edge. In general, cameras withsmallest pixelsto considerhigher resolutionimages while cameras withlarger pixelsthey have a larger surface area for collecting photons and producing themmore sensitive.
Most microscopes have optical output ports between 18 and 25 mm in diameter. Thus, without magnification (1x objective), the image would cover 18 to 25 mm of the sample. Given a fixed image size, camera sensors with a diagonal larger than the microscope's camera port will have pixels where no light is incident. That's why it's important to matchfield of viewcamera at maximum valuefield of viewmicroscope.
Larger pixels have a positive effect on sensitivity. Indirectly, they also have the benefit of the total time it takes to transfer the information to the computer. The total read time depends on the architecture of the camera, with CMOS being faster than CCD, but also on the total number of pixels in the camera. In general, a camera with more but fewer pixels will be ready for the next exposure faster than a camera with more smaller pixels.
Different microscopy methods detect specific interactions between light and the sample. methods than imagescatteredopreoccupiedluzlight focusto the sample using a separate illuminating lens and imaging objective. The focusing illuminating lens is known ascondenser, with appropriate working distance properties, NA, etc.
Fluorescence microscopy uses reflection orepifluorescencegeometry in which the lens serves as the illumination condenser and imaging lens. The illuminating light passes through the lens, and the detected light passes back through the lens and is divided into the camera or eyepiece. The advantage of this approach is that light that does not interact with the sample is directed away from the detector, maximizing the separation between the illuminating light and the fluorescent emission. The transmission and epifluorescent light paths are shown in Figure 4.
Two ways of lightingcriticaloKohler, are commonly used to illuminate a sample under a microscope. The main difference is that they copy the structure (critical) or encode the structure (Kohler) of the light source on the sample. Since Köhler lighting is the most commonly used, it will be discussed in this article.
Köhler invented a focused illumination system that allowed the field size, power and illumination angle to be controlled while encoding the structure of the light source projected onto the sample. He used the property of the lens to transform the side structure for it into parallel rays. Placing the light source at the focal point of the lens transforms the luminous flux into uniform light rays on the other side, changing the natural structure of the source. The many points emitted by the light source get mixed up and travel in parallel rays after leaving the lens.
Placing the light source close to the sample limits the control of the light intensity and field of view of the illumination. Köhler took pictures of the light source at the focal length from the condenser lens as shown in the figureFigure 5. This ensures control over the lighting field, using, among others,field stopin the center of the image component and aopening stop1f from the capacitor. The opening limiter is a very important design aspect; allowing easy control of the light intensity falling on the sample. These stops typically have levers that allow the user to manually adjust the area of illumination (field stop) and power (iris) delivered to the specimen.
There are many differentLamps,light emitting diodes (LED)ylaserswhich can be used to illuminate the sample under the microscope. Typical lamps used for lighting include:
- Halogen lamp. They provide broad-spectrum lighting and their output power is related to the voltage across the filament. Often used for broadcast images.
- xenon arc lamp. It has uniform power across the most commonly used wavelengths. In a light bulb, an electric arc flows through two metal points in a high-pressure xenon atmosphere, creating a plasma near the metal points. Sometimes used for fluorescence imaging.
- Mercury lamp/metal halide lamp. It has a higher total output at commonly used wavelengths than xenon lamps. It also generates plasma by an electrical discharge between two metal poles. Although the power at different wavelengths can vary dramatically, mercury vapor lamps are often used for fluorescence imaging. Cool between uses.
The useful life of each of these sources varies from:several hundred hoursfor mercury lamps1000-2000 hoursfor mercury/metal halide and halogen lamps.
LED light sourcesthey are powerful enough to compete with themxenonymercury/metal halide lampsas a light source in fluorescence imaging. Each LED has a unique color, which is why broadband LED sources come from arrays of many individual LEDs with a relatively narrow spectrum. LED sources have a lifespan of over 10,000 hours and are very energy efficient, making them very economical in long-term use. They can be quickly turned on and off in nanoseconds, making them useful for experiments that require tight lighting control. The spectral distribution of an example LED light source is shown in the figureFig.6.
lasersThey provide light with very specific wavelengths. For example, the light generated by ahel-neon (HeNe)The laser has a color of 632.8 nm. Unlike the other light sources discussed here, lasers provideconsistentlight. Coherence indicates that the light is highly ordered, with all the peaks and troughs of the light wave occurring at the same time and place. Coherence is necessary in focusing light to a diffraction-limited point, but it also complicates wide-field illumination due to its tendency to positive and negative interference. This self-interference can often be detected as a speckle pattern in the extended laser beam.
FiltersThese are optical elements that can transmit certain wavelengths of light while reflecting others. Color selection is critical in fluorescence imaging. An example of optical filtering is shown inFig.7.
Filters are commonly named because of their naturetransferand the wavelength at which they change from transmission to reflection, as shown in the figureFig.8. to 500 nmshort-pass filter (SP).it would transmit blue light with a wavelength greater than 500 nm and reflect red light with a wavelength greater than 500 nm. For comparison, 500 nmlong pass filter (LP).It will transmit light with a wavelength greater than 500 nm, reflecting light with shorter wavelengths.
Combining the properties of the SP and LP filters,band-pass (BP) filters.Were made. A 550nm SP filter combined with a 500nm LP filter would only pass light in between500-550 nm. BP filters are generally described by their center wavelength and allowed wavelengths on both sides. The hypothetical combination of the SP550 and LP500 filters is commonly known asBP525d25, BP centered at 525 nm with 25 nm transmission allowed on both sides (Reichman, 2017).
Under the fluorescence microscope, the combinationBP excitation filter, ALP dichroic filteriBP emission filterthey are housed in a cube-shaped holder to deliver high-intensity excitation light to the sample and effectively isolate the emission light from being directed into the chamber.
The parts of the microscope discussed here work together to send light into the sample, take light from the sample, and amplify it to a detector for collection. Aperture stops, usually found on the objective lens, limit the resolution of the microscope. Field limiters limit the illuminated or detected area. To get the best possible image, things like lenses, light sources, filters, and cameras need to be considered.
Abramowitz, M. 2003 Basics of the microscope and more, Olympus America, Scientific Department.
Davidson, M. W. Koehler Illumination on the Zeiss BasicResources website (https://www.zeiss.com/microskopia/us/solutions/reference/basic-microskopia/koehler-illumination.html)
Parry-Hill, M.J., Vogt, K.M., Griffin J.D. and Davidson, M.W. How to match the resolution of the camera to the microscope on the MicroscopyU website (https://www.microskopia.com/tutorials/matching-camera-to-mikroskop-rozwiązywanie)
Reichman, J. 2017 Handbook of Optical Filters for Fluorescence Microscopy. Chroma Technology Company Bellows Falls, Vermont (05101-3119https://www.chroma.com/sites/default/files/HandbookofOpticalFilters.pdf)
Spring, K.R., Parry-Hill, M. & Davidson, MWGeometric Construction of Ray Diagrams on the Olympus Microscopy Primer website (https://www.olympus-lifescience.com/en/microskop-resource/primer/java/components/characteristicrays/)
Spring, K.R., Parry-Hill, M., Burdett, CA, Sutton, R.T., Fellers, T.J. y Davidson, MW Laser Fundamentals on the Olympus Microscopy Primer website (https://www.olympus-lifescience.com/en/microskop-resource/primer/lightandcolor/laserhome/)
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