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Optical Microscopy

date2025-05-11tags:meche:

https://micro.magnet.fsu.edu/primer/techniques/index.html

Papers Three-dimensional Shape recovery from Image Focus using polynomial regression analysis in optical microscopy Sung-An Lee & Byung-Geun Lee DOI 10.3807/COPP.2020.4.5.411

Three-dimensional surface microtopography recovery from a multifocus image sequence using an omnidirectional modified laplacian operator with adaptive window size. Yingzhong Tian, Huijuan Hu, Haoyang Cui DOI 10.1364/AO.56.006300

github.com/usnistgov/MIST

Optical Table Using Bicycle inner tube

In many cases equipment is set up on vibration dampened tables. In microscopy, these optical tables, in the shape of a giant metal floating breadboard are ubiquitous, and expensive. These optical tables cost several thousand dollars from the cheapest suppliers. However, if microscopes are not mechanically isolated, oscillations attenuate the quality of images.

There’s an old trick if you can’t afford an optical table. Set your equipment upon an optical breadboard placed on an inflated bicycle inner tube. This is precisely what is demonstrated in a 2019 paper which details the creation of an affordable single-molecule localization microscope written by Hong Mao, Robin Diekmann, Hai Po Liang, Victoria Cogger, David Le Couteur, Glen Lockwood, Nicholas Hunt, Mark Schüttpelz, Thomas Huser, Vivien Chen, and Peter McCourt. https://www.degruyter.com/view/j/nanoph.2019.8.issue-7/nanoph-2019-0066/nanoph-2019-0066.xml

In the paper, they show that an inner tube at a relative pressure of 0.5 bar placed between an aluminum breadboard and a table significantly reduced vibrations, as shown by the repeated localization of the center of bright, fluorescent beads of 100 nm diameter.

Incu-Stream: Inverted Bright-Field Microscopy and Automated Mechanical Scanning for just $184

Incu-Stream is an open-hardware microscopy setup designed for bright-field cell imaging of incubated microplates. The design is all laid out in a paper written by Güray Gürkan and Koray Gürkan, with nothing hidden in a supplementary packet this time. https://ieeexplore.ieee.org/stamp/stamp.jsp?arnumber=8706880

The system is specifically aimed at creating a microplate compatible inverted bright-field microscope system that can be used in incubators. To come up with the outrageous price of $184, they use a low-cost CMOS image sensor, an inverted varifocal CCTV lens and an array of light emitting diodes.

The paper includes a table of used items and costs, circuit diagrams and pictures of assembly. If you are looking into building this device, the Github has CAD files for easy 3D printing, PCB layout files for easy circuit creation, and software. https://github.com/GurayGurkan/Incu-Stream

Since the device is intended to be used inside an incubator, it does lead us down the rabbit hole of DIY incubator design. Which is maybe the most common piece of equipment we see actually built in the labs we work in, and something we have never covered…

Optical Microscopy Illumination Sources

The performance of a light source in optical microscopy is determined by its emission characteristics (wavelength distribution, stability, brightness, and spatial distribution/coherence), the source geometry, and the optical components (lenses, mirrors) of the collector system (focal length, magnification, numerical aperture). The controllability of these parameters is crucial.

Common Light Sources:

Tungsten-Halogen Lamps

These incandescent lamps provide a continuous spectrum across the visible range, but much of the energy is lost as infrared heat. They are reliable and commonly used for various imaging modalities. The halogen gas reacts with evaporated tungsten, redepositing it on the filament, extending lamp life and improves brightness.

Mercury Arc Lamps

High-pressure mercury arc-discharge lamps are significantly brighter than tungsten-halogen lamps (10-100x), and provide intense illumination at specific wavelengths (when used with filters). Historically popular for fluorescence microscopy, they require careful alignment, have shorter lifespans, and pose potential hazards.

Mercury arc lamps, more so than other optical microscopy illumination sources, require a burn-in period after ignition in order to reach thermal equilibrium. They are also subject to flickering and are sensitive to their electromagnetic environment as well as their power supply.

Xenon Arc Lamps

Unlike mercury lamps, xenon lamps offer a relatively continuous and uniform spectrum across the visible range, with a color temperature (~6000 K) close to sunlight. This makes them suitable for quantitative fluorescence microscopy.

Metal Halide Arc Lamps

Metal Halide lamps are increasingly used in fluorescence microscopy. They combine an arc discharge lamp with an elliptical reflector and a liquid light guide. Advanced models include internal filter wheels, shutters, and neutral density filters. Their emission spectrum includes pressure-broadened mercury lines and a higher continuous background. [6, 9, modified]

Light-Emitting Diodes (LEDs)

LEDs are a promising technology for microscopy, offering advantages that traditional lamps lack (low voltage, battery powerable, switchable power). They offer diverse spectral outputs, making them suitable for exciting a wide range of fluorophores across the UV, visible, and near-infrared regions. High-power LEDs are now bright enough for many fluorescence applications. [7, 11, modified]

The wavelength of light emitted by an LED is determined by the semiconductor materials used in the p-n junction.

Light Source Power Considerations:

Choosing the correct light source depends on the illumination method (transmitted or epi-illumination), specimen characteristics, microscope setup, detector, and (in fluorescence) the fluorophore, filter sets, and acquisition speed. High illumination power is often acceptable for fixed cells with synthetic fluorophores or quantum dots. Live-cell imaging typically requires lower power levels. [8, modified]

Key Concepts Related to Light Sources:

Related to brightness, coherence affects the degree of interference in the image. Incoherent sources limit interference to the focal plane, while highly coherent sources can produce artifacts from dust and imperfections in the optical path. [7]

In some fluorescence microscopy light sources (e.g. with metal halide arc lamps) there is often incorporated an elliptical collection mirror. It is critical to position the arc correctly in relationship to this elliptical reflector for optimal formation of a focused beam into the microscope's optical train.[7,9]

The special housing for a mercury arc lamp contains the lamp, a reflector, a collector lens, and the electrical connections. Advanced lamphouses may offer automatic alignment and intensity control.[7]

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