How to Select and Configure a Microscope for Translational Medicine Research

DMi8 and Translational Medicine Research
- How to Choose and Configure a Microscope for Your Research The

new coronavirus may be one of the most dangerous viruses known to mankind, with strong transmission, hidden pathways and great harm, and facing such a formidable enemy, thanks to The development of nucleic acid detection technology can make it invisible
.
However, nucleic acid detection technology first needs to amplify the signal in nucleic acid.
As described in the latest version of the National New Crown Guidelines, the collected DNA needs to be expanded for 35 cycles before it can be used for detection.
The previous 40 loops are reduced, but the magnification is also as much as 2 ³⁵ , and it is conceivable that if there is any error in the middle, it will be magnified by such a high multiple
.
How can we measure it accurately? Standards are one of the most critical guarantees
.


Facing the standard products in the nucleic acid detection scheme of the new crown, Academician Fan Chunhai of Shanghai Jiaotong University has made the most critical contribution in this regard
.
At the beginning of 2020, with the active support of the National Center for Translational Medicine under his chairmanship, the joint team of Shanghai Metrology Institute and Jiaotong University worked day and night to develop the national nucleic acid reference material for the new coronavirus in just 42 days.
GBW(E)091111 and GBW (E) 091112, which was approved on March 19, 2020 by the national emergency authorization, provides quantitative traceability and quality control basis for nucleic acid kits for use by more than 100 research and development units, and makes important contributions to epidemic prevention and control.
Basic guarantee
.
Leica Microsystems, with 170 years of optical technology precipitation, is also very honored to provide support to the research of Academician Fan within its capacity
.

The picture is reproduced from the public account of "Shanghai Science and Technology", author: Yinuo .
If you want to do a
good job, you must first sharpen your tools.
Before achieving great scientific research results, the choice of equipment configuration is one of the primary factors
.
Optical microscopes are often one of the main pieces of equipment in life science research laboratories
.
It can help solve scientific mysteries in a variety of applications
.
Therefore, the configuration and functionality of the microscope is critical to the range of its applications, from brightfield microscopy to fluorescence microscopy and live-cell imaging
.

 What sample are you studying?
When choosing a research microscope, the primary consideration is the type of sample being studied
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For fixed samples placed on thin glass slides, you can choose an upright microscope
.
Live-cell assays require the use of special capabilities of the microscope because they are placed in large cell culture vessels filled with cell culture fluid
.

 Figure 1: Left: A slide on which a fixed sample, such as a tissue section, is placed
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Right: Petri dish for cell culture
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Only an inverted configuration with the objective below the sample and the condenser above the sample provides the essential free space and proximity between the objective and the sample
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At the same time, inverted microscopes also provide good access to cells, such as adding micromanipulators
.

Furthermore, living cells need the proper environment to survive
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Temperature and carbon dioxide concentration must be maintained at certain levels
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Accomplishing this task requires a climate chamber with corresponding controls
.

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Figure 2: Left: Upright microscopes feature an objective above the sample and a condenser below the sample
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Right: The inverted microscope is set up in reverse, primarily to give the user more space and the required proximity between the objective and the sample
.

 
In what dimensions do you think it is?
The microscopic sample unfolds into three dimensions: length, width and height
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Some samples, such as tissue sections, are imaged only in the xy direction, while other applications may require acquisition in the z direction as well
.
For imaging 3D volumes, e.
g.
for live cells, it is recommended to use a motorized turret, which is able to gradually guide your sample through the focal point
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Imaging software should be able to reconstruct a single image for 3D visualization
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For live cells, you have to add a time dimension
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In this case, system stability is another key characteristic
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Since temperature changes can affect the imaging system during acquisition, effective countermeasures must be taken
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Autofocus adjustments such as Adaptive Focus Control (AFC) eliminate these thermal effects and always find a predefined focus point
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 Figure 3: Adaptive Focus Control (AFC) can also automatically stabilize microscope focus during long time-lapse acquisitions
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The sensor detects the movement of the LED beam (850 nm), which can occur if the coverslip carrying the sample changes its position, for example due to thermal activity
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Which comparison method is best for your sample?
Most cells studied with microscopes—especially animal cells—do not have enough intrinsic contrast to allow researchers to see details
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The researchers used a contrastive approach to address this question
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Phase contrast (PH) and differential interference contrast (DIC) manipulate the light passing through the sample to increase contrast, and you can also stain it with fluorescent dyes (immunofluorescence) using fluorescent proteins, respectively
.

Depending on the contrast method, microscopes require specific equipment; for example, phase contrast requires special objectives, while DIC uses certain prisms that must be switched into the light path
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For fluorescence microscopy, you need special fluorescence filter cubes to force the correct wavelengths of light into and out of the sample
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 Figure 4: A series of neuronal cells obtained with different contrast methods
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From left to right: What about brightfield, DIC, phase contrast, fluorescence
 
light sources?
The choice of contrast method also determines the light source
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Transmitted light illumination for conventional brightfield microscopy, phase contrast and DIC can be performed using halogen or LED illumination
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Fluorescence microscopy can be performed with LED illumination or with the help of mercury, xenon or mercury metal halide lamps
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Do you want to record or publish your results?
If you want to take images of samples or perform live cell imaging, you need a digital microscope camera
.
Especially in the case of fluorescent live cell imaging, it is recommended to use a sensitive camera to minimize the amount of excitation light that could damage the cells
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In addition to mature CCD and EMDDC cameras, today's sCMOS cameras are also used due to their high quantum efficiency and acquisition speed
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For more information on digital microscope cameras, read the article "Introduction to Digital Camera Technology"
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Additionally, the large field of view (FOV) helps find regions of interest faster and image more cells simultaneously
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The camera port of a modern research microscope has a 19mm FOV, which is a perfect match for a 19mm sCMOS camera chip
.

Often it is not enough to just take a sample image, the data you obtain also needs to be analyzed
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To this end, easy-to-use imaging and analysis software facilitates quantitative data acquisition and reliable data analysis
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Do you need (3D) information to acquire thick samples?
Thick samples are a challenge for microscopy
.
Especially with widefield microscopy where the entire sample is illuminated at the same time, additional light from out-of-focus regions can significantly reduce in-focus sample features
.

Image resolution can help obtain images without out-of-focus light
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The technique can be applied to a single image plane for immediate results (ICC: Imaging Resolution Immediately), or can be combined with an additional deconvolution step (SVCC: Small Volume Imaging Resolution; LVCC: Large Volume Imaging Resolution) for better results
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Deconvolution redistributes photon information to their origin, providing better desired structural contrast in the focal plane
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This allows users to more easily distinguish structures of interest from the background compared to traditional widefield images
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 Figure 5: Single molecule RNA-FISH in cancer tissue
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RNA-01 (green), RNA-02 (magenta) Left: raw data
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Middle: resolved using instant imaging
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Right: After high-volume imaging resolution
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Courtesy of Prof.
Andreas Moor, University of Zurich (Switzerland)
.

 
Do you want to manipulate cells on a microscope?
Image processing of samples has become increasingly common over the past few years
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This means that researchers not only have to observe living cells, but also manipulate them with the help of light
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Fluorescence recovery after photobleaching (FRAP) is an example that helps to understand dynamic cellular processes
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For these types of manipulation techniques, additional light sources are often required, which must be integrated into the light path of the microscope
.

This method is not easy
.
The Leica Infinite Port is a universal solution to couple additional light sources into the light path of the microscope without compromising image quality, e.
g.
: FRAP, optical switching, ablation or optogenetics
.
With the right adapter, researchers can even connect their home-made devices
.

 Figure 6: The Leica WFFRAP module (black frame on the left) can be connected to the inverted research microscope Leica DMi8 via the infinity port
.

 
What is your budget?
An important question is how much money you can put in
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Some microscope suppliers offer predefined configurations for special applications
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But what if you don't need all the paid preconfigured components? This is why configuring components for free is more economical than purchasing a pre-defined microscope system
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Furthermore, the requirements for the microscope may change over time
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In this case, an upgradable system has certain advantages
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With predefined and fixed configurations, you will find that only a limited number of applications are required
.
Upgradability gives you the flexibility to configure according to changing needs
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With these factors in mind, modular microscopy platforms such as the Leica DMi8 allow researchers to start with an economical microscope system and then continuously upgrade as their needs grow
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 Figure 7: With its modular design, the Leica DMi8 can be configured according to the researcher's needs
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In addition, if requirements change, upgrades can be made at a later stage
.

 
Who uses microscopes?
The range of microscope users can vary widely
.
Especially in college, users can be very experienced or absolute beginners
.
Therefore, easy-to-use microscope systems run by intuitive software such as Leica Application Suite X (LAS X) help people get started quickly and acquire data quickly
.
For example, workflow-oriented design, image analysis wizards, and the seamless integration of peripherals into the system can simplify your work
.


In addition to widefield research microscopes, stereo microscopes are also frequently used in life science research laboratories
.
You can learn more in the article "Factors to Consider When Choosing a Stereo Microscope"
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Learn more: Leica Microscopy