White Papers

Camera Test Protocol

To ensure your camera is performing as well as it should be, we have designed a range of tests that can be performed on any microscope. The results of these tests will give you quantifiable information about the state of your current camera as well as providing a method to compare cameras, which may be valuable if you’re in the process of making a decision for a new purchase.

Why Gray Levels are Not a Quantitative Measure of Signal

Comparisons between the perceived brightness of biological features are often made based on the graylevel intensity values of the pixel. However, this can lead to potential misreporting of intensity values. Intensity should ideally be reported as a photon or electron count.

When comparing images received from different cameras, either for comparison studies or when assessing new cameras for purchase, comparing the number of graylevels reported in a biological feature can lead to misleading comparison data or the purchase of incorrect scientific equipment.

Control of Noise and Background in Scientific CMOS Technology

Scientific CMOS (Complementary metal–oxide–semiconductor) camera technology has enabled advancement in many areas of microscopy imaging. However, this technology also poses problems that camera manufacturers need to solve to produce a device capable of accurate quantitative imaging. 

To achieve this, several features of CMOS sensors have to be understood and then corrected. In this white paper, we’ll be focusing on the control of noise and background.

Increasing CMOS Camera Sensitivity Through Back-Illumination

Quantum efficiency is the measure of the effectiveness of the camera to produce electronic charge (electrons) from incident photons, where a higher QE results in the conversion of more photons to electrons of signal. 

This white paper will focus on how it was made possible to increase sensitivity on CMOS cameras by increasing QE to an almost perfect, 95% through the process of back-illumination.

Resolution

Resolution in fluorescence microscopy is defined as the shortest distance between two points on a specimen that can still be distinguished. This is primarily determined by two factors; microscope resolution, which is the smallest object the microscope can resolve, and camera resolution, which is the ability of the camera to detect what the microscope can resolve. 

Maximizing Microscope Field of View

Microscope field of view (FOV) is the maximum area visible when looking through the microscope eyepiece (eyepiece FOV) or scientific camera (camera FOV), usually quoted as a diameter measurement. 

Maximizing FOV is desirable for many applications because the increased throughput results in more data collected which gives a better statistical measurement for detecting subtle effects and also decreases time needed at the microscope.

Using Additional Optics to Adjust Pixel Size

Additional optics such as magnification couplers (also known as camera mount adaptors) can be used to optically adjust the effective pixel size and field of view of scientific cameras to better match the microscope resolution and field of view, respectively. Couplers are placed just before the camera on the camera port of the microscope and are available in several magnification or demagnification values.

The Effect of Camera Cooling on Signal to Noise Ratio

Cooling is a necessary feature on scientific CMOS cameras. Cooling directly reduces dark current, lowering the noise floor, as well as minimizing the occurrence of hot pixels. An uncooled scientific camera would not only struggle with low-light detection but, due to hot pixels, would also not perform as a true quantitative measurement device. 

On an uncooled camera, hot pixels would otherwise need to be controlled by interpolation filters which can be problematic for some applications requiring quantitative pixel uniformity such as in super-resolution localization microscopy.

Live Particle Tracking

Single molecule tracking grants researchers the incredible ability to observe molecular interactions and behaviorsat the single molecule level. This allows researchers to directly observe how molecules move, organize and travel, collide and react, associate and dissociate, often live and with high spatial and temporal resolution. The ability to do this provides valuable insight into live cell biology.

PrimeEnhance Active Image Denoising

Using an algorithm invented at INRIA and optimized for fluorescence microscopy in collaboration with the Institute Curie, PrimeEnhance implements a 2D denoising process which evaluates and processes incoming images to reduce the effects of photon shot noise at low signal levels. 

The algorithm also preserves the finer details and features of biological samples, and does not introduce image artifacts. One key facet of PrimeEnhance is the quantitative nature of the algorithm, ensuring that intensity values remain unchanged. 

PrimeLocate Data Reduction Technology

The PrimeLocate algorithm is most appropriate for localization microscopy methods such as STORM and PALM. The hallmark feature of localization microscopy is that sparse images of individual point emitters blink at random times during an image sequence. 

By finding the centroid of each emitter’s diffraction limited spot in a given frame and combining the localization results from each frame, a super-resolution image of the original fluorescence can be reconstructed.

Correlated Multi-Sampling

Two of the main components of read noise are “reset noise” and “amplifier noise”. Reset noise is a random offset created when a pixel is cleared of previous charge and can largely be eliminated using Correlated Double Sampling (CDS). 

With reset noise largely eliminated by using CDS, amplifier noise becomes the dominant source of read noise. A special implementation of CDS, called Correlated Multi-Sampling (CMS), can be used to reduce this noise contribution even further.

Programmable Scan Mode

Programmable Scan Mode provides increased control over the rolling shutter exposure and read-out functionality of CMOS sensors. The rolling shutter read-out behavior is a common implementation on CMOS sensors, and Programmable Scan Mode provides access to the sensor timing settings to allow optimization around imaging requirements.

A Review of eXcelon™ Technology

This paper provides a basic overview of the advantages and disadvantages of various types of low-light CCDs/EMCCDs and introduces a new sensor technology, eXcelon, that promises to mitigate some of their inherent limitations. 

Advanced Feature Set for the Evolve® EMCCD camera series

Teledyne Photometrics is continually developing new camera performance capabilities that streamline user workflows while enabling researchers to concentrate directly on the image data that is important to their studies. 

These advanced functions enhance the quantitative nature of the camera while simultaneously allowing scientists to analyze very specific data. All of the Advanced Features found in this document are available with every Evolve camera.