WIDE ANGLE
IMAGING - my heart and souls alike!
These days with the prevalence of work from home we are all pretty much used to sipping our morning tea or coffee and working on our laptops, attending virtual meetings while gulping down our breakfast or lunch – no need to worry, the audio and video are switched off! Believe me, I was introduced to this life from the very day I got my license to handle the confocal microscope-fidgeting with the stage controller and focus knob of the TPC with my left hand and setting the stack with my right. All of a sudden a reminder would tick in my mind.. Oh, I need to sip my coffee, it’s getting cold…Oh my God…it’s already 2 pm, the West canteen will be shut for lunch.
Yes, the confocal microscope which all of you are familiar with, but poor me, being a former microbiologist previously had the notion that only microbes can be observed through a microscope. It took me really some time to be convinced that one can view something else other than microbes under a microscope.
Nevertheless, getting a confocal microscope license at TIFR requires a lot of blood and sweat and so acquiring it at the end at least managed to bring a smile on my face and confidence in my heart. I have finally crossed one of my hurdles at TIFR (the upcoming one was clearing all my basic courses!) and managed to get access to the machine myself without the supervision of any senior or even my own PI. This itself was an overwhelming moment for me.....I was super excited but at the same time scared to go up to that machine with my own samples to image- is this machine going to be a devil or an angel in my life?
In my immense excitement and thrill to handle the scope all by myself, I was unaware that my samples, the Drosophila embryos, were helplessly drowning on a heavy layer of Vecta shield. So when I placed them on the stage and viewed them through the eyepiece, I was having a tough time in desperately trying to orient the embryos in the position where they required to be imaged. But I was cheering in my mind to see the embryos floating in the ocean of Vecta shield. Remember, I had no previous experience of orienting embryos, moreover, I had no clue as to which position I need to orient them for getting the image of my interest. Thanks to a very kind senior of mine who took all the pain and trouble to explain to me that I need to orient all the embryos dorsal up, and of course he had to teach me how I need to place them in that appropriate position with the help of a fine paintbrush. Well, never being a painter or photographer myself, it was a delight for me to simultaneously get hold of a paintbrush and an instrument to click pictures.
Now comes the next challenge, how to set up a Z stack? How to understand which slice to be the first one and which to be the last? Which area to focus on? The focus knob of the TPC and the joystick of the stage controller were like a steering and a brake to an untrained driver like me. If I am trying to focus at the anterior portion of the embryo, then my steering is driving me to the posterior end. I had even harder times when the embryo completely went out of the screen. Alas! I again need to adjust the field of view. There you go, even if I managed to get my ROI (I presume you all know what it means, ‘region of interest’ in the language of scientists!) into focus after several unfulfilled attempts, how to decide on the stacks? I went deeper and deeper till I saw nothing on the screen and again came upwards till the time I could see the drops of Vecta shield on my coverslip. Yes, that was the first image that I had set up a stack for all by myself, 150 slices, it went on for about 45mins (I don’t exactly recall). When I later on sat down to process and analyse that image, I was too taken aback to see that the first 50 slices were all black, then a few 10 slices of the embryo extremely bright and colourful, then all of the sudden, the embryo depicted a bit of ballet dance as it turned upside down. Yes, you guessed it right, it drifted. But I was overjoyed to have captured an interesting event, maybe thinking that the embryo has survived the fixation process or become alive with the laser flashing on it, knowing that it’s time for it to pose and show off. Apart from this, I was too happy to have all the embryos so brightly coloured in the images, oblivious of the fact that they were supersaturated. To me, it was as if photographs look beautiful when they are bright, then my images need to be bright if they have to look beautiful and pretty.
Anyway, I even showed those images in my first lab meet where all my seniors were too good and polite not to tell me anything else other than just saying ‘Modhura, your images are hitting our eyes, please see to it that they don’t get saturated…’ Truly speaking, it disheartened me a bit at that moment but later on when I became aware of my mistakes I couldn’t stop laughing at myself.
After that, the confocal microscope became my bread and butter for my entire project where I had to image not only stage fixed embryos but had to make long movies of about 1.5 hrs in almost every confocal slot. Gradually I lost count of how many embryos I had imaged in real-time for my M.Sc. project. Yah, it finally became imaging with my heart and soul; the machine and that small room became an extremely comfort zone for me. I jokingly used to say to myself, ‘Oh lord, I am finally living my M.Sc. life through live imaging!!!’