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Practical Guide to Choosing a Microscope Camera

W. Gray (Jay) Jerome Vanderbilt University School of Medicine , 1161 21st Ave. South , Nashville , TN 37232

Abstract: Current versions of image capture devices for light microscopes, CCD and sCMOS cameras, possess competitive but different specifi cations for several important parameters: pixel size, frame rate, quantum effi ciency, spectral response, dynamic range, and noise. While CCD sensors have been traditionally selected for resolution and sCMOS for speed, recent advances in both systems have blurred some of the distinctions. Also discussed are extreme low-light-level sensors and sensors for high-speed photomicrography. It is recommended that the fi nal selection be made on the basis of the performance of the camera systems on your microscope and for your application.

Introduction T e basic design of a light microscope has not changed much in the last several decades. How we use them, however, has changed dramatically and continues to evolve at a rapid rate. In part this evolution is driven by improvements in components that have allowed microscopists to approach imaging in new and creative ways. T e evolution of various super-resolution approaches to microscopy, for instance, are based on radical reconceptualizations of image information collection and analysis [ 1 ]. Some new approaches have been made possible by improvements in certain microscope components. One key component that has undergone rapid evolution is the image capture system. Although we still call these systems cameras, they are a far cry from the fi lm-based cameras with which many of us started our careers. In fact, current high-end capture devices constitute a group of hardware and soſt ware components adapted for specifi c types of microscopy. T ey have even shattered our defi nitions of resolution, since standard defi nitions of microscope resolution were based on theories that assumed the detection device would be the human eye. Many of today’s detection devices can outperform at least certain aspects of human vision by orders of magnitude. T ese advances in technology have provided a plethora of image capture devices and allowed new approaches to capturing image information. Super-resolution microscopy is but one of the fi elds that has been made possible by advances in image capture technology [ 2 ].

Although these advances allow microscopists to ask a much wider array of questions, they also necessitate the judicious choice of systems adapted to specifi c needs. In this rapidly evolving fi eld, it is very diffi cult to adequately review all possible choices. In general, however, image capture in the microscope can be divided into two collection schemes—one in which an entire fi eld of view is captured simultaneously and the other where image information is collected sequentially by illuminating the specimen point-by-point and reconstructing a two-dimensional image from the individual data points. Laser scanning confocal microscopes are an example of the latter. Each approach to image data capture has distinctly diff erent detector requirements. In this article, we will limit our


discussion to simultaneous image collection systems that do not discriminate wavelengths (that is, monochrome cameras).

Materials and Methods Digital image capture . Simultaneous image detectors are solid-state electronic devices composed of arrays of discrete photodetectors. Each detector captures photons coming from its respective area of the specimen and converts this information to an electrical signal that is proportional to the amount of light sensed by the detector. T e electrical signal is converted to digital information that can be stored or displayed. T ere are some key points regarding this process. First, the digital information is discrete and limited. Individual values must be in whole multiples of a single quanta of information; whereas, the analog information coming in to the system can have any real number value. T us, information is lost during the analog to digital conversion. Understanding what information is lost and knowing how to minimize the loss is a critical aspect of modern microscopy. T e second key point is that the capture device is divided into discrete sensors that correspond to the pixels of the acquired image. T us, the size of the individual discrete photodetectors helps set the limit of how small an object can be detected; more on this later. CCD and CMOS . Most microscope photodetector systems have some form of charged coupled device (CCD) or comple- mentary metal oxide semiconductor (CMOS) as the image sensor. Although there are a number of similarities, there are also dramatic diff erences between these two approaches [ 3 ]. T e CCD contains a two-dimensional array of photodiodes ( Figure 1 ). During image capture, charge builds up in each photodiode (pixel). Image capture is then ended, and the information is read out sequentially. T ere are various schemes for reading out, but essentially the charge cloud of each pixel is transferred one at a time to an amplifi er that measures the value of the charge and converts it to a voltage. Associated electronic devices then reduce noise, digitize the values, and output the digital value for storage in computer memory. T e main drawback of the CCD design is the sequential transfer time, which limits how fast individual images can be acquired because all pixel charge transfer processes must be completed before a new image can be collected.

Cameras employing CMOS technology also have a two-dimensional array of photodetectors that convert photons to electrical charge in a manner proportional to the photon energy absorbed. However, on a CMOS chip the amplifi - cation and digitization steps happen at each individual sensor in parallel before the information is passed off the chip for storage ( Figure 2 ). T is parallel processing approach dramati- cally reduces the time it takes to readout image information and prepare the sensor array to capture another image. One of

doi: 10.1017/S155192951700061X • 2017 September

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