FLOW CYTOMETRY
- When a sample is being introduced to a flow cytometry, it will first pass through the fluidic part. Explain in detail what happen to the sample from fluidic until the analysis of the sample. (20 POINTS)
Flow cytometry is the automated measurement of physical, chemical and biological properties of individual cells or particles flowing in a single stream in a fluidic system. The flow cytometer consists of three core system which are fluidics, optics and electronics. Before the sample enter the flow cell, the cells are moving in random and not in a single cell hence if the cells are close to each other it will results into the coincident events in which two cells are being analyze by the flow cytometry at the same time. Therefore, making the data obtain is less reliable and not accurate.
The accuracy of data of flow cytometry rely on the first system which is fluidics in which it is responsible in ensuring that all of the particles within the system are focused as a single cells and moving with same velocity. In fluidics system, it transports the sample from the sample tube to the flow cell. Once through the flow cell and pass the laser, the sample is either sorted or transported to waste. In order to make sure that the particles in the samples are in single cell and move in same velocity, a hydrodynamic focusing is being utilized in fluidics system. The hydrodynamic focusing system uses liquid to force cells to travel at the same speed and as a single cell. In hydrodynamic focusing, a sheath fluid is used to carry and align the cells or particles so that they pass through a narrow channel (core stream) with same velocity. Once the cells are focused into the core stream, the sample will then travel through the interrogation point (point in which the cells and the laser light interact) before either going to waste or being sorted. Therefore, this hydrodynamic focusing allows the analysis of one cell at a time by laser interrogation. The pressure of the sheath fluid sets the speed of the system. The differential pressure between the sample and sheath fluid can be altered according to the preferences of the rate of the cells flow passing the laser interrogation in a given period of time. Increase in pressure will increase the rate of cell flow and results in faster analysis. However, it may cause the core stream to be widen, allowing more cells to pass through the flow cell which will lead to the cells are no longer line up in a single file hence the resolution of the analysis may be loss. Therefore, to generate the most accurate data, the cells is run at a low concentration and as slow as possible because the slow rate will maintain a tight core stream, which will minimize coincident events and provide the optimal precision of signal produced.
After the fluidics system, the optics system will takes place. The optic system consists of various filters, light detectors, and the light source, which is usually a laser. This optic system involves a focused laser where they scatter light and emit fluorescence that is been filtered. The components of the optical system emit different wavelengths of light onto the cell, collect the data in the form of emitted photons and convert these photons to an electrical signal (photocurrent) that will go into the electronics system later. When a single cells flow from the fluidic systems, a light from a laser interrogates a cell causing the light to be scattered in all directions. This scattered light is then travel from the interrogation point down to the detector. The key parameters being measured by flow cytometry is the forward light scatter (FSC), side light scatter (SSC), and fluorescence emission signals. Light that is scattered in a forward direction which is the same axis as the laser travelled is called forward scatter. The intensity of this signal has been attributed to cell size and refractive index which is its membrane permeability. On the other hand, side scattered light (SSC) is light that is refracted by cells and travels in a different direction than its original path. It usually provides information about the granularity and complexity of the cells. Cells with a low granularity and complexity will produce less side scattered light, while highly granular cells with a high degree of internal complexity will result in a higher side scatter signal. Thus, by using forward and side scattered light detection, cell populations can often be distinguished based on characteristic differences in cell size and granularity. As well as separating cells based on FS and SS, cells can also be further separated by whether they express a particular protein. In this case, a fluorochrome is often used to stain the protein of interest. Fluorochromes used for the detection of target proteins emit light when excited by a laser with the corresponding excitation wavelength. These fluorescent stained cells or particles can be detected individually. Forward and side scattered light and fluorescence from stained cells are split into defined wavelengths and channelled by a set of filters and mirrors within the flow cytometer. The fluorescent light is filtered so that each sensor will detect fluorescence only at a specified wavelength. Filters are used in flow cytometry because of the different wavelengths of light are scattered simultaneously from a cell. Therefore, filters are needed to split the light into its specific wavelengths in order to measure and quantify them independently. Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting others. There are four types of filters which are long pass filter (transmit all wavelength above a specific wavelength), short pass filter (transmit all wavelengths less than specified wavelength), band pass filter (transmit a specific band of wavelength) and dichroic filters (long pass or short pass filters that contain a mirror coating).
Once the different wavelength of the light being emitted, detectors is used to capture the photons that are emitted by the excited fluorophores and scattered laser light, and convert them into photocurrent which is then passed to the electronics system. There are two types of photodetectors used in flow cytometry which are the photodiodes and photomultiplier tubes (PMT). Photodiodes is used for a strong signal such as the FSC detector meanwhile PMT is used to detect small amounts of fluorescence emitted from the fluorochromes and more sensitive than photodiodes.
Once the detectors collect photons of light, the electronic systems take place in which it converts the photons to electric current and travels to the amplifier where it is being amplified either using a linear or log amplifier which will then being converted into the voltage pulse. The intensity of the voltage can be adjusted by amplifying it on a linear scale or converting it to a logarithmic scale. The use of a log amp is beneficial when there is a broad range of fluorescence as that may need to be compressed and linear amplification is used when there is not such a broad range of signals. A voltage pulse is created as cells pass through the laser. As the cell passes into the laser, an event window opens and photons are emitted hence making the intensity of the voltage measured increases. More light is scattered as the cell moves into the center of the laser (maxima). As the cell leaves the laser, less and less light is scattered. After a set amount of time, the window closes until another object enters the beam and this leaves a pulse of voltage over time. The voltage pulse has three aspects; the pulse height is the maximum peak of light collected. The full pulse width is the time from the start of the pulse to the end of the pulse. The pulse area is the integral of the height over width (time) in which it will correlate directly to the intensity of fluorescence for that event. This pulse is then converted to a digital number and is transferred to the computer and becomes the data that will be analyse. All of the data associated with each individual cell are stored in digital data file that can be read and analyzed using the appropriate analysis software.
Flow cytometry data analysis is built upon the principle of gating. Gating is in essence electronic window that sets upper and lower limits on the type and amount of material that passes through. It is used to separate a sub-population from heterogeneous population. In analyzing the flow cytometry data, the data for each event is plotted independently to represent the signal intensity of light detected in each channel for every event. This data could be visually represented in multiple different ways and the most common types of data graphs used in flow cytometry include histograms, dot plots or density plots. Histogram is a graphical presentation of single-parameter data and shows the relative number and distribution of events. In histogram, the horizontal axis corresponds to the signal intensity of the selected parameters while the vertical axis represents the number of events (counts). Therefore, histogram tells the number of events (or cells) at a particular measurement value. Next, a dot plot is a graphical representation of two parameter data where each axis represents the signal intensity of one parameter. Each dot represents an individual cell analyzed by the flow cytometer. The characteristic position of different cell populations is determined by different physical properties such as cell size and granularity. Small to medium cells with low internal granularity will produce a medium forward-scatter and low side-scatter signal intensity where as a large cell with high internal granularity produces high forward- and side-scatter signal intensities. Density plot on the other hand is a graphical representation of two-parameter data where the colors represent the collection of events with the same intensity and each axis represent the signal intensity of one parameter.
- What is threshold? Explain how threshold affect the data you’ve obtain (5 POINTS)
A threshold is the lowest signal intensity value an event can have for it to be recorded by the cytometer. When a threshold is set, only the amount of light that surpass the threshold will be detected and form signals. The threshold can be set on any parameter but it is usually set on FSC. Setting a threshold could affect the data obtained as it only recorded the signal intensity more or equal to it only. This helps in reducing the background noises signal caused by the debris or cell fragments. This debris can overwhelm the data file and hence with threshold being set, a much more manageable file size and analysis will be able to be performed. Other than that, thresholding increases the percentage of target events in the data file by removing the undesired events. Hence the data obtain will be focused towards the cells of interest and removed the unwanted cells in our file. Therefore, the data file will only contain things we are interested in and decreases the file size. On top of that, it will make the data obtain more reliable as the data are all the event that we are interested in. However, it is needed to choose an appropriate threshold that is not too high as the data might be lose. This is because if we increase the threshold, anything beneath that will be invisible to the cytometer, and we will never acquire that data. Therefore, the threshold need to be set to its lowest value to ensure the entire desired events are being captured. However, the setting the of the threshold should not be very low as it will results in the noise becoming visible and interfering with the data analysis.
Microscope plays a vital role in healthcare field especially in the diagnostic lab. Microscope are used to view the cellular structures of organs, bacteria, germs and viruses which are too small to be seen by the naked eyes. With the advancement of technology, the microscope is now available in digital manner and brings lots of advantages to the users. With the availability of digitalized microscope, it helps to improved accessibility in which it allows users to be able to view and shared the slides anytime and anywhere via personal computer, tablet, or even smartphone devices. This is very helpful in diagnostic lab as it allows more health profession such as physicians to be able to see the slides image rather than they need to come to the lab to see the slides by themselves.
Moreover, the used of digital microscope in diagnostic lab helps to minimize errors since all of the processes such as the tissue sections or staining in pathology lab and the reports are in one place hence the chances to accidentally exchange a slide or a report is eliminated as the image is automatically linked to the correct report via a unique number or a barcoding. In addition, slides can no longer be lost and the images can be retrieved again with digitalized microscope. Other than that, digitalized microscope makes the workflow of the diagnostic process more efficient and faster. This is because all of the requests, slides and reports are automatically linked together making it more efficient. Physical slides which is usually stored in archived that is often have to be searched takes a lot of time especially when it is needed for discussions to diagnose a disease. With digital microscope, with only one click to the computer, all of the images and report of the slides are stored and can be retrieved easily making it to be faster and efficient method to be used in diagnostic lab.
Furthermore, digitalized microscope makes it possible for the users to capture entire slides which is impossible in the conventional microscope. Even with low magnification in the conventional microscope, it is hard to see all the area of the slides at once hence making the users to miss something and increases the potential of seeing the same part of the slides and making incorrect analysis for the diagnosis. Therefore, with the availability of the digitalized microscope we can minimize this mistakes as the entire slides can be seen at once and it allows the users to keep track on what they are seeing through the computer screen. In addition, digital microscope gives a long-term cost benefits as it eliminates the costs of physically shipping the slides from one site to another. It also can minimize the cost to replace a breaking and loss of the glass slides as replacing the glass slides can also be costly.
Other than that, the images form by the digitalized microscope is in high resolution. It helps the user to have a better view of the slides. Digitalization enables the user to have a multiple angle views and zooming the slides making it more efficient, easier and faster for diagnostic approach. The digital output can be magnified on the monitor hence making it easy to do a closely examination on the slides. On top of that, digital microscope allows a live observation hence making it possible to conduct any real-time test and diagnostic procedure. Real time images allow visual progression of life cycles, observation of a specimen over a period of time and the ability to return to a particular image for the diagnosis processes.
Digital microscope also allows the users to improved analysis as it allows measurement to be directly done by the software. Measuring is much more accurate on digital images than on physical slides, and can also be done in easily manner. As a result, the diagnostic process becomes more transparent and making the process of diagnosing a disease to be faster. For an example, an automated image analysis tools can be used to help in the interpretation and quantification of biomarker expression within tissue sections.
The advantages of connecting the computer to the microscope is that it produces digital images in which this digital images can be shared among the peers. This is helpful in diagnostic procedure in which it enables the health profession to be able to exchange the images for any second opinion or consultation from the other specialist. This can be done in easier manner hence making the discussion on a certain rare cases for diagnosis to be more efficient. Additionally, the images can be shared easily amongst physicians during task shifting. The images also can become part of the electronic patient record hence all the specialist even the patients are able to view the images easily. On top of that, it also able to show up to four images side by side viewer hence making it easier to make any comparison during the process to diagnose a disease.
Next, digitalized microscope also helps to save space in the diagnostic lab itself. This is because it does not need to have any physical space for storage of the slides. All of the slides images can be saved directly into the computer and can be retrieved anytime when needed hence making the bench space to be minimized because all of the process are in one system. On the other hand, the digital slide however can be stored forever especially for rare cases of diseases for future reference and the quality is still the same as compared to the glass slides which may breaks or fade over the time. On top of that, the digitalized microscope helps to reduced turnaround times as it enables a faster access to archived digital slides. Reduction in the time required for slide retrieving, data matching, and organizing results has significantly reduced the turnaround time compared to manual processes hence making it more efficient for diagnosis to be done if the slides are needed to be retrieved.
Lastly, with the digital microscope, the neck and back pain problems of the users can be reduced due to the ergonomics of the instrument. This is because the image of the sample is displayed on a monitor directly and the users can analyse it while sitting in a comfortable and relaxed position. This is really important in the diagnostic lab as high sample are needed to be deal with, making the person working with the microscope feels tired when they use the conventional microscope. Therefore, digitalization of the microscope helps the user to be able to work longer and stay more focused.