Quantitative Bio-element
Imaging Center (QBIC)
BIF MICROSCOPY WORKSHOP
MARCH 25TH, 2015
DIRECTOR: PROFESSOR THOMAS V. O’HALLORAN
MANAGING DIRECTOR: KEITH MACRENARIS, PH.D
History and Mission of CLP and QBIC

“The Chemistry of Life Processes Institute acts as an umbrella for a variety of centers,
facilitates collaborations and helps bridge different cultures. By lowering the barriers to
scientific discovery, the Institute hopes, for example, to design new drugs for the
treatment of cancer and neurodegenerative diseases as well as develop improved
techniques for diagnosing diseases earlier.”

Designed to be a collaborative, interdisciplinary effort to map the “inorganic signatures of
life”

Started in 2003 and established as an official center within the Chemistry of Life Processes
Institute in September of 2008

CLP includes 39 tenure-track faculty members and 11 cores and centers

Elemental Analysis is located in Silverman Hall East Room B540

Microscopy is located in Silverman Hall East room
Organization of Imaging Facilities within CLP
How to Map the Inorganic Signatures of Life
Elemental Analysis
Imaging
Collaboration
Using high end instrumentation
for quantitative metal analysis to
determine metal concentrations
in solutions, materials, cells and
tissues.
Using one-of-a-kind STEM, 2-photon
laser scanning microscopy, and
laser ablation ICP-MS for elemental
mapping in tissues and cells
The synergy with Argonne
National Laboratory and the
Advanced Photon Source for
Hard
X-ray
Fluorescence
Microscopy for subcellular trace
elemental mapping
QBIC Space in Silverman Hall
1571
Upright Confocal Microscopy
Meta Detector
Objective Turret

First instrument installed was Upright Confocal
microscope in 2003 with manual tunable
spectra physics 2-photon laser

Zeiss LSM 510 Upright Confocal Microscope

Equipped with Argon (488 nm), 2 HeNe (545,
633 nm) continuous wave lasers

Beginning in September 2008 a softwaretunable Spectra physics MaiTai DeepSee
Ti:Sapphire 2-photon Laser

Equipped with a tunable Meta Detector for
multi-wavelength and spectrum analysis
capabilities

Short working distance high N.A. objectives
[10x, 20x, 40x, 100x (oil)] as well as a 63x, 0.9
N.A. dipping lens
Two-Photon Excitation Phenomena

Simultaneous absorption of two photons in order to excite
a molecule from a ground state (usually) to an excited
electronic state.

Generally uses a femtosecond-pulsed solid state near IR
laser such as Ti:Sapphire du to low probability of nearsimultaneous absorption of two photons hence the need
for high flux.

With two photons absorbed simultaneously the probability
of fluorescence emission increases quadratic ally with
excitation intensity.

This restricts excitation to a small focal volume (~ 1
femtoliter)

Advantages: Minimal phototoxicty and sample damage
due to the use of NIR-IR light, increased penetration
depth (up to 1.5 mm), and tunable excitation
wavelength increasing the library of usable fluorophores
for laser scanning microscopy.

Major disadvantage: Tendency to produce heat due to
plasmonic excitation in nanoparticles particularly gold.
Emission Ratiometric Imaging of Intracellular Zinc
Excitation @ 710 nm using 2-photon laser
No treatment
+ 10 µM zinc sulfate
J. Am. Chem. Soc., 2004, 126 (3), pp 712–713
+ 1 mM TPEN
Inverted Confocal Microscopy

Zeiss AxioObserver.Z1 Inverted confocal
microscope with LSM 510

Installed in September of 2008 with
software-tunable Spectra physics MaiTai
DeepSee Ti:Sapphire 2-photon Laser

Equipped with Argon (488 nm), 2 HeNe
(545, 633 nm) continuous wave lasers

Equipped with 3 PMT detectors and
Discovery V8 Pentafluor & stage top
incubation

Short working distance high N.A. objectives
[10x, 20x, 40x, 63x (oil)]

X-Cite Fluorescence Illumination System
and DIC optics for 20x and 40x objectives
Nanoparticle Imaging using Confocal Microscopy
(Left) A fluorescence micrograph of Gold
nanostars (AuNS) loaded with high densities of
nucleolin-specific DNA aptamer (Apt-AuNS).
(Right) Fluorescence micrographs of 6 different
cancer cell lines incubated with Apt-NS. (Odom
Group, Chemistry)
Mouse (SKH1-E) skin treated topically
with 1∶1 Aquaphor only (Left) or with 50
nM
Cy5-labeled
(red)
SNA-NCs
dispersed in the 1∶1 Aquaphor (Right).
(Paller Group, Dermatology)
Microinjection/Micromanipulation

Olympus IX53 Inverted microscope installed June
2014

10x, 20x, and 40x long working distance Hoffman
printed objectives for high resolution imaging in
plastic

Equipped with an Eppendorf Transferman NK2
microinjection system, including a Warner
Instruments Picoliter Injection System

Equipped with a Color CCD camera for image
acquisition and analysis

Computer workstation with CellSens Software
High Speed Fluorescence Microscopy

Olympus IX83 Inverted Fluorescence Microscope
installed June 2014

Motorized z-stage with zero drift compensation

Equipped with a 30 fps ANDOR Zyla sCMOS
camera with 5.5 megapixel format and 22 mm
field of view

Long working distance DIC/Phase condenser
(N.A. 0.55, W.D. 27 mm)

2.5x, 10x, 20x, and 60x (oil) long working distance
objectives

Motorized fluorescence turret with filters for
DAPI/FITC/TRITC/Cy5
CCD versus CMOS

Both convert photons of light into electric
charge and process it into electric signals

Charge Coupled Devices (CCDs) every pixels
charge is transferred through a limited number
of output nodes and sent off as an analog signal

Complementary Metal Oxide Semiconductors
(CMOS) is designed to have every pixel to have
its own charge to voltage conversion and
includes amplifiers, noise correction, and
digitization circuits where the chips output is in
digital bits

Generally, CCDs have lower noise and more
uniformity with better signal

CMOS cameras have very high frame rates
Laser Ablation ICP-MS
STEM/EDS in NUANCE
Information

For Microscopy and LA-ICP-MS email: Keith MacRenaris at
keithmacrenaris2009@u.northwestern.edu

For STEM/EDS analysis email: Reiner Bleher at bleherreiner@gmail.com

QBIC website: http://qbic.facilities.northwestern.edu/

NUANCE website: http://www.nuance.northwestern.edu/