hello everyone in this video I will discuss about in details the principles of confocal microscopy and several imaging parameters now if you watch my previous video I hope you would understand why we should use confocal microscopy why it is a little bit better than fluorescence microscopy what is the basic principle behind it and in which cases a confocal microscope could be used in this video I would cover the following topics I would cover why confocal over fluorescence I would also cover optical sectioning property and see stacking adjusting gain and offset and several other imaging parameters in order to image optimally also scanning modes used to scan the laser scan the sample with the laser also detectors used in confocal microscopy and what all parameters determine the image quality and resolution so stay tuned this is one of my favourite microscopes this is a Zeiss confocal microscope here you can see the main part the main confocal 'ti lies in this scan head so the scan it holds a pinhole and the detector so this is the important part if you get rid of them then the rest of the microscope is nothing just like a HEPA fluorescent microscope and one of the reason to do confocal microscopy is to get these beautiful images but that's not actually true here are two images two same images which are taken in a epifluorescence microscope and a confocal microscope clearly you can tell in hepa fluorescent microscope there are a lot of backgrounds as if the image is like fuzzy and blurry image also another image you can see the same the confocal image is way more sharp and crisp and the resolution is better in case of the confocal microscope image and the reason being conformed uses laser whereas epifluorescence uses normal light of single wavelength so since the laser has a property of coherence the laser can be focused into a very narrow point known as laser waist whereas the waist is way bigger in case of heavy fluorescence so the chance that less amount of fluorophore would be activated at a time in confocal is high but in case of epifluorescence quite a lot of fluorophores in the surrounding regions would be also activated and that give rise to that blood now Clearasil here is a fluorescence microscopy diagram so we give a excitation light and let's say our sample has Fitzie or gfp anything so we are sort of exciting it with a 488 wavelength light and we're collecting the emission in a camera so the fluorophores that we are targeting would be effect would be kind of excited and we would collect the emission light but the furo force of upper and lower plane of that image would be also excited and also it would enter the camera that would result in a blurry image whereas in a confocal in the same excitation paradigm with a laser we're collecting the emission line and since there is a pinhole the pinhole is income the pinhole is conferred with the specimen plane that means the specimen plane and the pinhole are in same focus so only focused light can pass so here is another light ray which is coming from above or below plane of that imaging plane so it cannot pass through the pinhole and reach the detector thereby we don't getting we are not getting the stray lights and that's why we are the quality of the image is actually increasing also the resolution now we would talk about the optical sectioning and sales taking property of the confocal system so confocal system comes with a motorized stage so the distance between the objective lens and the stage could be altered in real time and thereby it can at a time image several focal planes and after that all the local focal planes could be marched to reconstruct a 3d image of the object now if I give you example it would be way clearer so you can see here the the objective is moving up and down or the stage is moving up and down main point is the distance between the objective and the stage is changing and thereby you're going through the image plane York you're going all around the image plane and imaging all the planes later a 3d reconstruction of the image could be possible like this so we are actually not physically sectioning whereas we are at a time imaging from a particular focal plane and we are collecting light from a particular focal plane so that give rise to these beautiful images another important point and most important point to get good confocal images is to how to optimally adjust gain and offset first of all what is gain and what is offset so gain means so gain is a simp simplistically gain is a parameter change in which we can increase or decrease the signal if we increase the gain simply the signal will increase later when why we would discuss the detectors we would understand the basis of increasing gain but gain mean increasing game means increasing voltage across the PMD electrodes offset means addition of a negative or positive voltage now if I give you example these things would be more clearer now here you can see this is the baseline for the multiplier signal so even if there is no specimen under a microscope there is some amount of signal coming to the detector due to some thermal emission of electrons due to other reason there are some kind of signal now by giving a negative voltage you can make that residual signal very close to zero and then you can increase the gain or increase the voltage across the PMT's across the electrodes of the PMT you can increase and boost the signal by changing gain now here is a image where you can see the gain here is increased too much that means way too much voltage is injected that's why you are seeing everything is saturated so this kind of paradigm is not proper another image you can see here it is pretty much offset but the gain is not increased properly that's why still the features here that we wanted to image are still dim so gain and offset need to be properly adjusted such that we get a proper dynamic range of detection and in a moment I would discuss what is dynamic range simply in an image if we increase the gray gain it would increase the signal but at the same time you see the image looks more grainy because the background signals also increase while we increase the gain now why does anyone need to adjust gain or offset so in order to utilize the full dynamic range of imaging a person need to utilize a person need to just the team and off set optimally here you can see there is an image and this image is quite proper an image that has all sort of great values would be called a good image so if you don't understand this image I have another image for you in this image the black black things or black or blueish kind of pixels are actually low valued pixels whereas a yellowish pixel is very high value pixel if you look at the image you would see all Nisour all sort of colors are present it's not that one color is present more than other colors this kind of all colors are blended properly so all sort of gray levels are present that means we have utilized the whole dynamic range and we have kind of imaged for the older values and captured all the features of it now I would show you three comparison here the image looks too saturated because if you look at these lookup table you can see there are too much white into it that means the image is too saturated in order to improve the image quality in this case you need to deduce gain or laser power here the dynamic range is kind of proper because all sort of gray values all sort of range range of pixel intensities are captured in this image whereas this image has way too low signal because you can barely see all the features inside this image so we are missing a lot of information from this image now I will talk about another important feature which also is important for a resolution of thoughtful image is the digital digital zoom apart from the normal zoom apart from the normal magnification that means for example we are increasing the magnification from 40 X - 60 x apart from that there is something called digital magnification or digital zoom so what happens in digital zoom let's say the laser is scanning we are at 40 X and the laser scan the hole filled wholesale depicted here when we increase the zoom then the laser would scan for a small feature in the sill so it's simply scanning the list area but the image that would be produced in the both the cases have the same size so more so more feature would be kind of displayed in case of when we increase the zoom for example let's say there are three pixels very close enough now if we increase the number of pixels by projecting the image into a bigger frame then there are more gaps between it that increases the resolution in other words in case of increasing zoom we are not changing the imaging size but a small subset of feature is projected onto the same number of pixels so pixels per feature is more such that the image quality gets better and also the resolution get better for example here is the image in 40 X now once we add a 3x digital zoom you can clearly see the image quality has improved and the resolution also looks way better now we should talk about the scanning modes and the type of scanner used in confocal microscope so definitely we use laser light sources for confocal microscopy so laser kind of scans in a small portion now question is how the laser raster's in a sample in a small spot so the answer is there are several kind of scanners that are used in the confocal microscope which are galvanometer based scanner and resonance scanner why different type of scanners are used galvanometer based canners use 2 meter which are perpendicular to each other and they are vibrating well there is a change in electrical field so Calvin Ramirez scanners are slow scanners they can scan at a rate of one to five frames per second whereas the resonance scanner are pretty fast scanner and they can scan 30 frames per second so in order to carriage fast dynamically life processes a resonant scanner would be a better option these days there is hybrid scanner which employs both galvanometer scanner and resonance scanner so those are known as also hyper scanners or a hybrid scanners next we would talk about the detectors used in confocal system con focus mainly used photomultiplier tubes or PMT's now here is the basic outline of a PMT so there is the incident light incident light is the emission light from the field of work and it would hit a photo cathode now this photo cathode is generally made by multi alkali or sometimes alloy like gallium arsenide phosphide now different different material can be used to build this photo cathode depending upon the difference in the material infused the photo cathode sensitivity could be also changed however when the light hit the photo cathode some electrons are ejected that electrons are actually going through successive anodes and ultimately reaches the impaneled and there there is another digital to analog to digital converter which is converting these signals and digitalizing this signal and thereby the image is reconstructed point by point now here is an example of two different kind of detector that could be used in case of multi alkali detector the problem is when the electrons are bouncing back and forth in the diode arrangement cathode the problem is there is a loss of electron and that means some amount of signal is also lost but in case of a hybrid detector special arrangement of the of the detector ensures that the the kind of the loss of the electrons are minimized so that's why hybrid detectors are better now sure I would talk about what determines or what factors determine the image quality of a confocal microscope simply the pixel dwelling time laser intensity pin hole diameter gain and offset floor for quality and detector quality all these factors are important in determining the confocal image quality so I would briefly go over to these points because in the next video I would discuss these points in details so pixel dwelling time means how much the laser is dwelling on a particular point so if the laser too is too much time there is a possibility of photo bleaching so you have to optimize the dwelling time laser intensity is another important factor if the laser intensity is too low the features won't be illuminated and if the laser intensity is too high then the features would be saturated and the dynamic range won't be fulfilled in the image the pinhole diameter is another important thing if the pin hole diameter is stool Neptuna or too less then what happened a lot of signal would be cut off and the image would be deep but if the pinnacle diameter is more then what would happen the out-of-focus light can get through this pinhole now also gain an offset or other parameters that are important determinant of image quality because I have already shown you with examples that if you properly if you don't add just gain and offset properly the image looks horrible and other than that forever is the most important if the staining or the fluorophore quality is not good or exam the fluorophore is pitched then you are never going to get a good image in confocal microscopy after that detector quality is important now we have to ensure that we are always imaging in a linear range in a linear range of the detector in order to get good images now definitely the hybrid detectors or kalium arsenide phosphide detectors are way better than the multi alkali detectors that's why choosing the proper detector for your system is also very important now if you liked my video give it a quick thumbs up don't forget to Like share and subscribe thank you