Staining and mounting are processes that enhance the contrast and visibility of your specimen, as well as protect it from damage and deterioration. Staining involves applying dyes or chemicals that bind to specific structures or molecules in your sample, making them stand out from the background. Mounting involves placing your specimen on a glass slide and covering it with a cover slip, usually with a drop of liquid medium between them. You should use staining and mounting techniques that are compatible with your microscope type and objective lens, and that highlight the features of interest in your sample. You should also avoid using too much or too little stain or mounting medium, as this can cause blurring, distortion, or fading of your image.

Buy a service contract and stay in touch with the company who sold you the system. Laser alignment deteriorates over time. In fact, all alignment deteriorates over time. You need to have trained personnel come in to maintain the system and ensure that it's performing according to specifications.

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This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?

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Try to utilize immersion liquids between the objective's front lens and the cover slip. Use objectives with 60x to 100x (or higher) magnification that designed for use with immersion oil.

As explained in the previous question, Koehler Illumination should be the standard protocol with each and every objective used. This solves all issues calibration remains unchanged if the lenses are kept clean and free of oil and dust. Common error is to use oil emersion and switch back to lower power lenses that then get coated or spotted with oil. The price is immediate loss of resolution and calibration. One thing I learned from Leica engineers is never to clean lenses with alcohol that leaves a coating behind as well as dissolving the lens mounts. Best thing is acetone but ask an engineer/expert to show you how.

The TE™ technology addresses the problem of performance longevity of ageing LEDs by making the exchange of the engine a simple procedure that can be carried out in just a few minutes. But transferable means so much more than replaceable!

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The unobtrusive T11 Fresnel is specifically designed to operate in any noise-sensitive environment, with TV studio use enhanced via Cpulse™ flicker-free software and a dedicated plus and minus green channel control.

Epass™: Ethernet pass through switch which sustains Ethernet integrity, when the fixture has no power, to automatically maintain network connectivity - on request

Image processing should be minimal. In fact, you should limit it to reducing the "shot noise" from the PMT's on the confocal. If in doubt, collect the PSF from your system and visually inspect the z volume. Remember to collect the data using sub nyquist zoom on your objective. Maybe 28x? Usually a sampling rate of 0.2microns in about 6 stacks on a 1micron bead. With theses parameters you'll easily see the specific PSF cone surrounding by flecks of noise accumulating mostly around the top edge of the cone.

Our AirLOC™ (Less Optical Cleaning) technology greatly reduces the level of airborne particles drawn over the optical elements.

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Image processing software is a tool that allows you to enhance, analyze, and manipulate your microscope images, using various algorithms and functions. Image processing software can help you improve your microscope resolution and clarity, by applying filters, adjustments, corrections, and transformations to your image. For example, you can use image processing software to sharpen, smooth, contrast, crop, rotate, resize, or annotate your image. You can also use image processing software to perform quantitative measurements, such as counting, measuring, or calculating parameters from your image. However, you should be careful not to overprocess or alter your image in a way that compromises its validity or integrity. You should also document and report any changes or modifications you make to your image, and use the original image as a reference.

Be careful with image processing. Do everything you can to get the best image from the microscope as described in other questions and then try and limit to basic functions of brightness and contrast. People get into trouble publishing over processed images.

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Optical Power Meters. An optical power meter (OPM) is a type of electronic test device used to measure the power output of fiber optic equipment or the power or ...

The Robe Ethernet Access Portal allows to access internal data from a networked fixture, viewed as a web page, addressable via the fixtures network IP.

Regarding what magnification should be used, ask yourself, what am I trying to demonstrate with this image? If you are looking to show a population of cells, a whole organism, or tissue, go with a lower mag, like 4X or 10X. In mammalian cells, if you are looking for subcellular details, neurite branching, filopodia, or working with smaller eukaryotic, or prokaryotic organisms go to 20X or higher magnifications. The type of lens (air, oil, water immersion, phase) you can use will ultimately be dependent on the type of microscope you are using. Not all lenses can be used on all microscopes. Choosing the right microscope/or imaging system for your imaging needs should also be considered.

Cpulse™ is a PWM (Pulse Width Modulation) control system for luminaires that allows you to select and fine-tune the LED driving frequency to ensure no flicker will be visible on any camera system.

With the MSL-TE™ 350W LED Engine generating over 10.085 lm from the Fresnel fixture and all the advantages of cost, exchange or replacement, this LED technology provides identical colour characteristics, responses, and control of T-Series luminaires, giving absolute colour consistency across your Robe lighting installation.

First use lower magnification to choose an area to be explored. Then use higher magnification oil immersion lens. Make sure the lens is cleaned after every use.

Building on the superb MSL™ (Multi-Spectral Light) colour concept, and innovative TE™ technology, our static T11 Fresnel™ maintains the exceptional colour finesse of the phenomenally successful T-Series. With a fast, interchangeable front lens system, the T11 Fresnel™ offers absolute, cost-effective flexibility while delivering superb optical quality.With the MSL-TE™ 350W LED Engine generating over 10.085 lm from the Fresnel fixture and all the advantages of cost, exchange or replacement, this LED technology provides identical colour characteristics, responses, and control of T-Series luminaires, giving absolute colour consistency across your Robe lighting installation.A virtually controlled CCT range of 2.700K to 8.000K (extended range 1.800K – 10.000K), calibrated whites, and tungsten emulation gives precise control, while we ensure skin tones are perfectly rendered due to T11’s high TLCI and TM30-18 ratings and exceptional CRI of 95+. Capitalizing on the benefits of TRANSFERABLE ENGINES to address the need for task-specific LED solutions, we introduce two additional TRANSFERABLE ENGINE options. The TE™ 350 LED DL (Daylight) White 6.000 K engine boasts a CRI of 97 and yields over 5.700 lm, while the TE™ 350 LED TGW (Tungsten White) White 3.200 K engine delivers over 4.500 lm with a CRI of 97, as measured in an integrating sphere.High-definition optics deliver the classically soft Fresnel style beam, with a wide-ranging 5-60° zoom range.Our innovative manual shuttering system, functioning as an internal barn door, is both accurate and easy to use. Fitted in the fixture is a manual 1° MagFrost™ for instant softening, with a 5° MagFrost™ stored on the magnetic holder within the fixture. Other optional frosts are available.The unobtrusive T11 Fresnel is specifically designed to operate in any noise-sensitive environment, with TV studio use enhanced via Cpulse™ flicker-free software and a dedicated plus and minus green channel control.Utilizing the rapid-change front lens system, the T11 Fresnel™ quickly transforms into a Profile or PC fixture for total luminaire flexibility without the cost of separate generic purchases.T11 The three into one generic solution!

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Maintenance is the best way! Focus on one kind instrument! Update the software! Update the hard ware! Communication with colleagues

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A virtually controlled CCT range of 2.700K to 8.000K (extended range 1.800K – 10.000K), calibrated whites, and tungsten emulation gives precise control, while we ensure skin tones are perfectly rendered due to T11’s high TLCI and TM30-18 ratings and exceptional CRI of 95+.

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The objective lens is the part of the microscope that collects and focuses the light from the specimen onto the eyepiece or camera. The magnification and resolution of your image depend largely on the quality and type of the objective lens you use. Generally, higher magnification lenses have higher resolution, but they also have lower depth of field and require more light. Therefore, you should choose the objective lens that best suits your purpose and sample, and adjust the light intensity and focus accordingly. You should also clean the lens regularly with a soft cloth and lens cleaner, and avoid touching it with your fingers or other objects.

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There are a range of imaging techniques that do no require staining but use the microscopes ability to use the properties of light and its interactions with the specimen. These include bright filed, dark field, phase contrast, and interference contrast. Different techniques can be used also including total internal reflection which is ideal to identify adhesion of cells to cover slips and culture wells, Not to mention extended depth of field microscopy, using light slits and passing the specimen through the light to give perfect focus of the whole specimen. Fluorescent microscopy can be used to localise auto fluorescent elements in specimens. These techniques yield a lot of information before deciding on staining techniques.

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Our innovative manual shuttering system, functioning as an internal barn door, is both accurate and easy to use. Fitted in the fixture is a manual 1° MagFrost™ for instant softening, with a 5° MagFrost™ stored on the magnetic holder within the fixture. Other optional frosts are available.

The DataSwatch™ inbuilt virtual colour library for Robe LED fixtures consists of up to 237 pre-programmed and calibrated colours and tones, allowing for rapid and accurate programming.

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Robe has included a dedicated +/- green control channel in fixtures that feature our MSL™ specific white TE™ source fixtures. This allows you to make the smallest changes of green content compared to the fixed levels produced by the specific filters on white source fixtures.

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Utilizing the rapid-change front lens system, the T11 Fresnel™ quickly transforms into a Profile or PC fixture for total luminaire flexibility without the cost of separate generic purchases.

MagFrost™ - magnetic paddle fast change system providing exchangeable frost containing as standard a medium 5° for even wash

10.085 lm, CRI 95+, + - Green Correction Function, Cpulse™ special flicker free management for HD and UHD cameras, ready for 8K and 16K up to 25.000Hz

Capitalizing on the benefits of TRANSFERABLE ENGINES to address the need for task-specific LED solutions, we introduce two additional TRANSFERABLE ENGINE options. The TE™ 350 LED DL (Daylight) White 6.000 K engine boasts a CRI of 97 and yields over 5.700 lm, while the TE™ 350 LED TGW (Tungsten White) White 3.200 K engine delivers over 4.500 lm with a CRI of 97, as measured in an integrating sphere.

Sample fixation and antigen umasking. Make sure that the samples are properly fixed right after collection. Thick tissues require longer incubation in fixative such as 4% PFA. If the frozen sections are desired, tissues need to be snap frozen as soon as possible. After sectioning, some staining may require antigen unmaskin or permeabilization.

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The General Device Type Format creates a unified definition for exchange of data for the operation of intelligent luminaries, such as moving lights. The file format is human readable and developed using open source formats.

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20241125 — We found the best overall light to be the Westcott 18" Bi-Color LED. We like that it has a digital readout for brightness and temperature and ...

Robe lighting’s Epass™ provides Ethernet in/out connections with a pass-through switch that sustains Ethernet integrity when the fixture has no power to automatically maintain network connectivity.

There is no need to be stuck with frosts supplied with the fixture! Robe’s MagFrost™ magnetic paddle system provides you with quickly exchangeable frosts, enabling you to select those most suitable for your production.

AirLOC™ (Less Optical Cleaning) technology greatly reduces the level of airborne particles drawn over the optical elements. This increases the overall performance, light quality and time between routine cleaning and maintenance.

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The condenser and diaphragm are parts of the microscope that control the amount and direction of light that reaches the specimen. The condenser is a lens that focuses the light from the source onto the specimen, while the diaphragm is a device that regulates the size of the aperture that lets the light through. By adjusting the condenser and diaphragm, you can optimize the illumination and contrast of your image, as well as reduce glare and artifacts. You should align the condenser with the objective lens, and use the diaphragm to match the aperture of the objective lens. You should also experiment with different levels of brightness and contrast to find the best settings for your sample.

Measure the original, NOT the processed image. The goal of image processing is to allow segmentation algorithms to correctly identify targets in the image. Segmentation masks are used to measure attributes of the associated target in the image itself. Inspect the segmentation mask produced to prevent introducing artifacts. Analysis pipelines with multiple processing steps also required more time and computation power to perform the operation. However, if you apply the final segmentation to the ORIGNIAL UNPROCESSED image, the results are valid. Ai-guided software can bypass preprocess steps needed for accurate segmentation, but results are still only as good as the training (ai-models can also be over trained).

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Building on the superb MSL™ (Multi-Spectral Light) colour concept, and innovative TE™ technology, our static T11 Fresnel™ maintains the exceptional colour finesse of the phenomenally successful T-Series. With a fast, interchangeable front lens system, the T11 Fresnel™ offers absolute, cost-effective flexibility while delivering superb optical quality.

Practice refractive index matching. For example, with oil immersion lenses the RI of the oil is 1.515. The sample should be mounted in mounting medium with RI as close to 1.515 as possible. Refractive index matching reduces spherical aberration, particularly at depth, and will give the sharpest images with the best resolution. This is critical for thicker samples.

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The calibration and alignment of your microscope are important factors that affect the accuracy and reliability of your measurements and observations. Calibration is the process of verifying and correcting the scale and dimensions of your image, using a standard reference object such as a micrometer or a calibration slide. Alignment is the process of ensuring that all the optical components of your microscope are in proper position and orientation, such as the eyepiece, the objective lens, the condenser, and the light source. You should check the calibration and alignment of your microscope regularly, especially if you notice any changes or inconsistencies in your image quality or performance. You should also follow the manufacturer's instructions and recommendations for maintaining and servicing your microscope.

Koehler illumination is an absolute essential to get the optimum resolution, free of artefacts with any microscope. In this the light is focused on the specimen and cantered in the the precise focus of the objective with the condenser aperture closed so that the circle of light fills 2/3rds of the visible field of the objective when looking down the eyepiece with lens removed. This cuts off the edges of the lens which tends to scatter light away from the focus. The image is then crisp and maximised for resolution with that objective. The procedure to obtain Koehler illumination should be automatic every time you change objectives.

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Alignment of the system is critical to maintain performance. If you find that you are bombarding your sample with laser emission I think it's time to call your service engineer. With modern day PMT's you should only be using about 0.5-1% laser power and that's even for antobodies/fluorophores to rare proteins.

... light rather than directing it into the lens barrel. The rugged X2 frames form a weather-seal on the lens and are built to withstand extreme wind, salt ...

To get the best possible image qualitty, ensure that the microscope is properly aligned. This includes aligning the condenser, the objective lens and using the correct diaphragm setting. The amount of light that enters the microscope is controlled by the diaphragm. A smaller diaphragm setting will increase the contrast of the image, but it will also reduce the resolution. You can do it yourself by following the instructions in the microscope manual or get a qualified technician align the microscope for you.

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Robe's MSL™ (Multi-Spectral Light) LED light engines are, specifically, designed to consistently deliver the highest quality light through the additive colour mixing method. The MSL spreads the emitted light equally throughout the Planck curve, thereby providing the widest possible colour gamut, while simultaneously delivering seamless full range CCT control.

In addition to the observations by colleagues. It is important to avoid dust settling on the lens and ocular lens. Always avoid scratching of the lens with the aperture and stage of the microscope. The cleaning of the lens before after usage is critical to maintain the clarity of the specimen image during the observation.

Microscopes are essential tools for laboratory management, as they allow you to observe and analyze specimens at high magnification and resolution. However, sometimes you may encounter problems with the clarity and quality of your images, which can affect your results and interpretations. In this article, you will learn some tips and tricks to improve your microscope resolution and clarity, and how to avoid common pitfalls and errors.

Optimize primary/secondary antibody concentration ratios to prevent over saturation during detection step. Don’t overexpose during image processing.

One has to optimize the different microscopic parameters according to the unique requirements of his/her experiment. Microscopic sample processing is not the same for every experiment, rather it greatly depends on the nature and type of the sample. I recommend using appropriate dyes and with proper and timely maintenance of your microscopes will help a lot in getting good and acceptable micrographs.