Patent ID: 11906433
Assignee: INSTITUTO POTOSINO DE INVESTIGACIóN CIENTíFICA Y TECNOLóGICA A.C.
Field: Measurement (Instruments)
Classification: CPC G | IPC G

Claim 0:
1. A method for three-dimensional imaging using bright field light microscopy, the method comprising the steps of:
a) placing an unstained sample on an optical transparent holder, surrounding the unstained sample in the transparent holder with air or an optically transparent liquid or solid, and then placing the sample in an optical microscope, the optical microscope having automated change of focus, a substage condenser fitted with an adjustable aperture iris diaphragm, a digital camera to record images of said unstained sample created by said microscope, and one or more digital computers to store and process the images of said unstained sample captured by said digital camera;
b) operating said optical microscope in bright field mode;
c) adjusting said optical microscope to Kohler illumination and operating said optical microscope under Kohler illumination;
d) adjusting the aperture of said condenser iris diaphragm of said optical microscope so that contrast is generated in the images of said unstained sample captured by said digital camera of said optical microscope;
e) acquiring a Z-stack of images of said unstained sample;
f) transferring the outputs of said digital camera to said computer or computers;
g) applying to said Z-stack of images a high-pass filter to remove the low spatial frequency components from images;
h) applying a pairwise frame subtraction operation to the resulting image stack of step g);
i) applying a smoothing filter to the resulting image stack of step h);
j) apply a white top hat filter in three dimensions to the resulting image stack of step i); and
k) reporting the result of step (i) in the form of digital image stacks, matrices of numbers, projection images or three-dimensional rendering images.