The Universe Within

Observation, manipulation
and fabrication of
microscopic objects

Vincent Ricardo Daria

Instrumentation Physics Laboratory
Microscopy, Signal Processing and Instrumentation

Co-prepared by: Dr. Carlo Mar Blanca

Objectives Fundamentals of microscopy

• Use of LIGHT to observe structures with sizes in the

1. Determine how magnifiers function. range of ~10-6 m

2. Learn the history and physics behind the microscope. EM Spectrum

x/gamma rays uv visible infrared microwave
3. Components of the microscope
decreasing λ 0.4 10-6 < λ < 0.8 10-6 Increasing λ
400 10-9 < λ < 800 10-9
4. Magnification and Resolution
2x10-3 m
0.1x10-9 m 10x10-9 m Integrated circuits
Fish
atoms protein Bacteria, plant/animal cells
5. Discuss the current and modern uses of microscopy. Red blood cells ~ 7x10-6 m
eggs
1x10-9 m E coli ~ 2x10-6 m
molecules

Ray tracing Image formation

Object
Image
Real
Images

F F
Inverted
Virtual
Magnified Images
Real
3 simple rules:
1. ray passing thru focus goes out parallel
2. ray passing thru lens center goes undeflected
3. ray going parallel passes thru the focus

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 Ray tracing – Magnifying lens Ray tracing - Microscope
Eyepiece
image (Virtual)
Eyepiece
Object

Objective lens Eyepiece

Upright Object
Magnified
Virtual Image
F0 Fe

F0 Fe
Object

Real Image from

the objective lens
Simple rules:
1. ray passing thru lens center goes undeflected Simple rules:
2. ray going parallel passes through the focus 1. ray passing thru lens center goes undeflected
2. ray going parallel passes through the focus

Ray tracing - Microscope History [1595 AD - ]

Eyepiece
1590 Zacharias
Jannsen
Real • inventor of the
microscope
Image
1700 Antonie van Leeuwenhoek 1635 Robert Hooke
• Simple, 300 X magnification • cork cells

Objective

Sample

Illumination

History [1595 AD - ] History [1595 AD - ]

Designer microscopes

Novelty microscope

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 History [1595 AD - ] Microscope components
Designer microscopes

Microscope components Microscope components

The most important optical component of the microscope
is the OBJECTIVE.
Microscope parameters
• Magnification
1. Gathers light passing through specimen and projects
an accurate, real, inverted IMAGE of the specimen into
the body of the microscope. • Resolution

Magnification
The second is the EYEPIECE. Eye Piece Objective
Magnification (10X) Magnification (60X)
2. Magnifies the objective image a second time as a
virtual image seen as if 10 inches from the eye to be
captured by the eye or a camera.
Microscope
The third is the SUBSTAGE CONDENSER.
× = Magnification

3. Gathers light from source and concentrates it in a collection of parallel beams

(from every azimuth) onto the specimen.

Objectives Image formation

Numerical Aperture (NA) Fourier
plane
f f f f
• NA is measure of ability to gather light and resolution Object, O(r) Image
+High Freq
NA = sinα
Ideal Case -Low Freq

• NA can be increased by increasing α -High Freq

small NA High
LARGE NA NA

Finite lens A
+High Freq
-Low Freq

α aperture -High Freq

+High Freq
B -Low Freq
-High Freq
Large NA, MORE LIGHT ! Low
NA

http://microscopy.fsu.edu/primer/anatomy/objectives.html

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 Objectives Imaging Applications
Spatial Resolution
• Radius of focused light is
R
R = wavelength
NA

Ascaris eggs Head louse

• Small R, → HIGH resolution

• Small wavelength → HIGH resolution
• LARGE NA → HIGH resolution
Human Flea Lonestar Tick

Cells like you’ve never seen them before Conventional microscopy

PSF=PSFdetection

Neural network of a
mouse embryo
(fluorescence image)

ftube lens

ftube lens
M=
fobj

Blur
fobj
Focus
Blur
Thick Sample

Wide field illumination

Confocal microscopy 3D Microscopy

Laser Confocal Fluorescence Image
Flourescence Microscope at different axial
positions of an eye
Pinhole Detector of a mouse embryo
Pinhole stained with DAPI

Source 60 µm 100 µm

λ=λa

Point
source
140 µm 180 µm
Axial
Resolution
Beam
High contrast image
Splitter
Objective (fluorescence image)
Lens

3D color coded Image of the back of a

y mouse embryo (stained with DAPI) showing
z the formation of somites

x Thick Sample V Daria, et al, “Proceedings of Scanning Laser Microscopy 19,” May 1998
V Daria, et al, “Image contrast enhancement for two-photon fluorescence microscopy in
turbid medium,” Applied Optics 37, p7960 (1998)

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 Mouse embryo 3D applications

Medaka embryo 4
days old

Lateral
Projection
Fluorescence imaging

• 3D images of developing embryonic

organs

• Sample E10.5 mouse embryo

HNF3β for endoderm
Neurofilament for nerve tract
Dorsal-Ventral
3D Reconstruction
Projection

Observing semiconductor integrated circuits Moving microscopic objects using optical forces:
Optical Tweezers, Arthur Ashkin, 1986
Optical trapping of a high index particle

Laser

Optical Beam Induced Current (OBIC)

V Daria, et al, US Patent Pending (PCT 00013)

V Daria, et al, High Contrast image of semiconductor
sites via one-photon optical beam induced current
imaging and confocal reflectance microscopy,
Applied Optics (2002)

Moving things with light Moving things with light

Optical trapping of multiple particles

Fixed arrays Interactive sorting Cell positioning

Size

PJ Rodrigo, VR Daria, J Glückstad, Optics Express 2004

VR Daria, PJ Rodrigo, J Glückstad, App Phys Lett 2004

Pre-programmed
movements
Color

Microgates
and turbines
Speed: 5 µm/s

R Eriksen, VR Daria and J Glückstad, Opt Exp PJ Rodrigo, R Eriksen, VR Daria and J VR Daria, PJ Rodrigo and J Glückstad,
25, 566 (2002) Glückstad, Opt Exp 25, 566 (2002) Biosensors and Bioelectronics (2004)

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The world’s smallest Bull / Micro-machines The world’s smallest Bull / Micro-machines

Kawata, et al, Nature 2001 S. Maruo, et al., APL 2003

y-scan
S. Maruo, et al., IEEE MEMS 2001 (2001)

Min Gu et al, Aus Opt Soc News, (2001)

x-scan

Satoshi Kawata, et al, Nature 2001

Observation of Small Scale

Structures

Thank you.